Inhibition of RecBCD Enzyme by Antineoplastic DNA Alkylating Agents

doi:10.1016/j.jmb.2006.06.068

J. Mol. Biol. (2006) 361, 898–919

Inhibition of RecBCD Enzyme by Antineoplastic DNA Alkylating Agents Barbara Dziegielewska 1 , Terry A. Beerman 1 and Piero R. Bianco 2,3,4 ⁎ 1

Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA 2

Department of Microbiology and Immunology University at Buffalo, Buffalo, NY 14214, USA 3

Department of Biochemistry, University at Buffalo, Buffalo, NY 14214, USA 4

Center for Single Molecule Biophysics, University at Buffalo, Buffalo, NY 14214, USA

To understand how bulky adducts might perturb DNA helicase function, three distinct DNA-binding agents were used to determine the effects of DNA alkylation on a DNA helicase. Adozelesin, ecteinascidin 743 (Et743) and hedamycin each possess unique structures and sequence selectivity. They bind to double-stranded DNA and alkylate one strand of the duplex in cis, adding adducts that alter the structure of DNA significantly. The results show that Et743 was the most potent inhibitor of DNA unwinding, followed by adozelesin and hedamycin. Et743 significantly inhibited unwinding, enhanced degradation of DNA, and completely eliminated the ability of the translocating RecBCD enzyme to recognize and respond to the recombination hotspot χ. Unwinding of adozelesin-modified DNA was accompanied by the appearance of unwinding intermediates, consistent with enzyme entrapment or stalling. Further, adozelesin also induced “apparent” χ fragment formation. The combination of enzyme sequestering and pseudoχ modification of RecBCD, results in biphasic time-courses of DNA unwinding. Hedamycin also reduced RecBCD activity, albeit at increased concentrations of drug relative to either adozelesin or Et743. Remarkably, the hedamycin modification resulted in constitutive activation of the bottom-strand nuclease activity of the enzyme, while leaving the ability of the translocating enzyme to recognize and respond to χ largely intact. Finally, the results show that DNA alkylation does not significantly perturb the allosteric interaction that activates the enzyme for ATP hydrolysis, as the efficiency of ATP utilization for DNA unwinding is affected only marginally. These results taken together present a unique response of RecBCD enzyme to bulky DNA adducts. We correlate these effects with the recently determined crystal structure of the RecBCD holoenzyme bound to DNA. © 2006 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: RecBCD; DNA helicase; nuclease; DNA alkylators; antineoplastic agents

Introduction Many anticancer agents bind covalently to DNA, introduce bulky adducts, and inhibit DNA metabolic processes including repair, replication and transcription.1–3 Frequently, the first enzymes to encounter DNA adducts are DNA helicases. These enzymes progress in a unidirectional manner through the DNA helix and unwind the duplex

Abbreviations used: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; SSB, ssDNA-binding protein. E-mail address of the corresponding author: [email protected]

producing nascent single-stranded DNA (ssDNA) in a reaction fueled by energy derived from the hydrolysis of nucleoside 5′-triphosphates.4–7 Consequently, if a bulky adduct disrupts helicase progression, this might translate into drug-induced inhibition of the repair, replication or transcription processes.8 Several DNA-binding drugs, including minor groove binders and intercalating agents, have been evaluated for their ability to inhibit DNA helicases in vitro. Several DNA helicases have been utilized as model systems, including mammalian helicase II, the Bloom's and Werner's proteins, simian virus 40 (SV40) large T-antigen (TAg), herpes simplex virus (HSV) UL9 and the Escherichia coli Rep, UvrD and RecBCD enzymes.8 These studies demonstrated that the level of drug-induced inhibition of DNA

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DNA Alkylators Inhibit RecBCD Enzyme

unwinding depended on both the agent used and on the DNA helicase being tested. For example, daunorubicin was shown to potently inhibit DNA unwinding by Tag and mammalian helicase II, but affected DNA unwinding by the bacterial UvrD and Rep helicases only moderately.9–12 Similarly, intercalating agents inhibit UvrD, whereas minor groove-binding agents do not.9 In contrast, whereas minor groovebinding compounds are potent inhibitors of both the Bloom's and Werner's DNA helicases, intercalating agents do not inhibit these DNA helicases significantly.13 Thus, the ability of a DNA-binding compound to inhibit a DNA helicase is a combination of the properties of the agent being tested and those of the DNA helicase(s) being studied. Although the agents alluded to above and those used in this study exert their effects in eukaryotic cells, the well-characterized RecBCD enzyme of Escherichia coli is an excellent model enzyme to study the effects of DNA-damaging agents on DNA unwinding. This is the best characterized DNA helicase to date, with a large body of both in vivo and in vitro data available.14 In contrast to other DNA helicases, the RecBCD enzyme is the complete nanomachine, requiring no additional proteins to mediate efficient DNA unwinding. Second, although the enzyme unwinds double-stranded DNA (dsDNA) non-specifically, it does read the sequence of the unwound DNA as it passes through the core of the enzyme. Thus the ability of an agent to inhibit sequence-specific regulation can also be evaluated using this enzyme. Third, in addition to being well characterized both in vivo and in vitro, the crystal structure of the enzyme in the presence of DNA was determined recently.15 Thus, it is possible to correlate the in vitro effects of DNA adducts on DNA unwinding with the three-dimensional structure of the enzyme. RecBCD is a combination helicase/nuclease that is critical to homologous genetic recombination and DNA repair in E. coli.14,16 The holoenzyme consists of the three subunits RecB, RecC, and RecD.14 The RecB and RecD subunits are NTP hydrolysis-dependent DNA motor proteins that drive DNA translocation and strand separation,17,18 while RecC functions in holoenzyme scaffolding, DNA unwinding and recognition of the recombination hotspot χ.15 Together, these subunits form a complex enzyme possessing DNA helicase, ATPase and nuclease activities. In vitro, RecBCD binds to blunt or almost blunt dsDNA, ends with >106-fold affinity relative to internal sites.19 The helicase unwinds the DNA at rates of up to ∼1000 bp s−1 at 37 °C, traversing on average 30,000 bp per binding event.20,21 DNA unwinding is accompanied by the simultaneous and asymmetric endonucleolytic degradation of unwound ssDNA with the 3′-terminated strand, relative to the entry point of the enzyme, being preferentially degraded (Figure 1(a)).22 For both translocation and DNA degradation to occur, RecBCD requires Mg2+ and the energy derived from the hydrolysis of a nucleoside triphosphate, typically ATP.23 The nuclease activity of RecBCD is

899 regulated by the recombination hotspot χ (chi for crossover hotspot instigator; defined as 5′-GCTGGTGG-3′).24 The translocating RecBCD enzyme recognizes χ as the unwound single strand of DNA but only when approaching χ from the 3′-side (Figure 1(b))25 Recognition of χ results in the translocating enzyme pausing at χ,26 during which the polarity of the nuclease activity is altered.27 The 3′ to 5′ activity is attenuated while the 5′ to 3′ nuclease activity is up-regulated. Consequently, continued translocation past χ results in RecBCD producing 3′-tailed ssDNA, onto which the RecA protein is preferentially loaded, thereby facilitating the initiation step of homologous recombination.28 In addition to controlling the polarity of the RecBCD nuclease activity, the encounter with χ results in an uncoupling of the two RecBCD helicase motors (RecB and RecD), so that DNA unwinding beyond χ is powered by the slower RecB helicase with the RecD motor having been inactivated at χ.26 Thus, a properly oriented χ-sequence controls the polarity of degradation and the rate of enzyme translocation. To understand more clearly how these activities are combined in the heterotrimeric enzyme and to understand more clearly how DNA alkylation might effect DNA helicase activity, we summarize the relevant features of the structure of RecBCD determined in the presence of DNA.15 The RecB and RecC subunits are intimately associated with one another, with the RecD subunit being more peripheral (Figure 2(a)). The RecB and RecD subunits couple the hydrolysis of ATP to DNA translocation and strand separation by pulling DNA into the holoenzyme, and through the RecC subunit, where strand separation occurs.17,18 Although RecC has no demonstrable enzymatic activity, it acts as a scaffold onto which the two motors assemble, it is intimately involved in strand separation and is responsible for χ recognition.29,30 Finally, the enzyme contains a single nuclease active site that resides within the C terminus of the RecB subunit, and which is positioned approximately opposite the entry point of dsDNA into the holoenzyme. Unwinding of dsDNA by RecBCD involves an intimate coordination between subunits, and is proposed to occur in several sequential steps, utilizing a quantum inchworm mechanism.31 First, the enzyme binds to dsDNA ends with the leading domain of RecB reaching out 23 nt ahead of the ssDNA/dsDNA junction. Here, it interacts with the minor groove of the duplex and initially anchors the enzyme in place (Figure 2).15,31 Thereafter, this domain translocates the duplex into the enzyme, where it is separated into ssDNA by RecC (Figure 2(b)). The unwound single strands of DNA pass through 11–16 Å diameter channels in RecC (one each to RecB and RecD; Figure 2(b), inset). Each motor translocates on the unwound single strands of DNA and, as a consequence, the unwound ssDNA is pulled through RecC to the nuclease active site, where it is subsequently cleaved. As RecBCD is intimately associated with the DNA during translocation and DNA unwinding, it is

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DNA Alkylators Inhibit RecBCD Enzyme

Figure 1. Unwinding of dsDNA by RecBCD and regulation by the recombination hotspot χ. (a) A schematic showing the unwinding of χ°-DNA by the translocating RecBCD enzyme. Although RecBCD can unwind this DNA substrate from either end, only one orientation is shown. Here, RecBCD translocates from right to left. The RecB subunit translocates along the 3′-terminated strand relative to the entry point of the enzyme (the top strand), while RecD travels along the 5′terminated strand relative to the entry point of the enzyme (the bottom strand). During translocation and DNA unwinding, the 3′-terminated strand is degraded more vigorously than the 5′-terminated strand. (b). Regulation of the nuclease activity of RecBCD by the recombination hotspot χ. The χ-site (5′-GCTGGTGG-3′) is present in the upper strand of the substrate, as shown (the 3′-terminated strand), and the arrow indicates the direction from which RecBCD must encounter χ in order to recognize and be regulated by this octanucleotide sequence. Before interacting with χ, the 3′terminated strand is degraded more vigorously. Upon χ-recognition, the enzyme pauses, and the polarity of degradation is altered so that, during subsequent translocation, only the 5′-terminated strand is degraded.

evident that bulky, covalently attached DNA adducts could potentially affect any or all of the following components of the unwinding reaction: first, disruption of the interaction of the leading domain of RecB with duplex DNA to delay progress of the duplex into the enzyme; second, the actual strand separation step could be affected by increasing the melting temperature of the DNA duplex; third, covalent attachment of bulky adducts could slow the progress of unwound ssDNA through the channels within RecC; fourth, adducts bound to specific sequences, such as χ, could affect recognition of χ as the unwound ssDNA passes through RecC and immediately before interacting with the nuclease domain of the enzyme, and fifth, the alkylation of DNA may perturb the allosteric interaction that activates the enzyme for ATP hydro-

lysis, manifested as an uncoupling of ATP hydrolysis from DNA unwinding. To ascertain how bulky adducts resulting from intra-strand alkylation might perturb the function of RecBCD, we selected three distinct DNA alkylation agents. The three agents, adozelesin, ecteinascidin 743 (Et743) and hedamycin, possess unique structures and sequence selectivity; they covalently attach bulky adducts and significantly alter the structure of DNA (Figure 3). These molecules bind to dsDNA and alkylate only one strand of the duplex in cis, and have been shown to inhibit DNA replication,32–34 and repair.35,36 Below, we present the relevant details of each these agents. Adozelesin belongs to the family of extremely cytotoxic, cyclopropylpyrroloindole anticancer

Figure 2. The RecBCD holoenzyme is associated intimately with the DNA. (a) RecBCD enzyme bound to dsDNA is shown from the top so that the relative distributions of each subunit and the DNA substrate (colored in orange) are readily discernible. The RecB subunit is shown in green, and the position of the nuclease domain is indicated. To visualize the path of the unwound single strands of DNA, RecC (light mauve) is transparent. The dsDNA substrate is split apart by the interaction with two methionine residues present in RecC (highlighted in fuchsia within the broken yellow circle). The RecD subunit is shown in brown. (b) A side-view of the structure is shown with the RecD subunit removed so that the ssDNA channel within RecC can be seen. The enzyme is rotated 90° relative to the orientation shown in (a). The leading domain of RecB protein is shown reaching out 12 nucleotides ahead to translocate the duplex DNA into the enzyme. The tail of one strand of DNA is shown passing through one of the two channels present in the RecC subunit (indicated by the broken yellow circle and the inset) and is in transit to the RecD subunit (not shown).

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DNA Alkylators Inhibit RecBCD Enzyme

Figure 2 (legend on previous page)

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DNA Alkylators Inhibit RecBCD Enzyme

Figure 3. Structures of DNA alkylating agents used in this study. (a)–(c) The chemical structures of the alkylating agents used. (a) adozelesin, a cyclopropylpyrroloindole containing three subunits: A, cyclopropyl; B, indole; and C, benzofuran. (b) Et743, a tetrahydroisoquinoline consisting of three fused rings, A–C; and (c) hedamycin, a pluramycin antibiotic containing planar an anthrapyranthrione chromophore with two sugar molecules attached at the corners and a reactive double epoxide.

agents that irreversibly and covalently associate with DNA in the minor groove. 37 Adozelesin consists of three subunits, A (a cyclopropyl unit), B (an indole unit), and C (benzofuran) (Figure 3(a)). It binds to DNA in the minor groove at PuPy/PuTTA sequences, and modifies the 3′ adenine via its cyclopropyl moiety.38 The interaction of CC-1065 with DNA, adozelesin's parent agent, has provided a model for the structural features of the adozelesin– DNA complex. Both CC-1065 and adozelesin bend and stiffen the double helix, increasing the melting temperature of the DNA by as much as 20 deg. C.39 On binding to DNA, CC-1065 widens the minor groove, due to the ethano bridges between the B and C subunits. As adozelesin has an amide linker at the same position, it is unable to widen the DNA minor groove to the same extent as CC-1065.40,41 Instead, adozelesin forms strong hydrogen bonds between the amide linker of the indole and benzofuran subunits, and the carbonyl group of the central thymine. Consequently, this positions the drug not centrally within the minor groove, but instead towards the modified DNA strand.40 As a consequence of DNA duplex distortion and alkylation, both adozelesin and CC-1065 have been shown to inhibit DNA ligase, DNA polymerase and DNA helicases in vitro.42–44 Ecteinascidin 743 (Et743) is an anticancer antibiotic isolated from the Caribbean tunicate Ecteinascidia turbinate. It is a carbinolamine-containing, DNAbinding agent consisting of three fused tetrahydroisoquinoline rings, the A, B and C subunits (Figure 3(b)). The A and B subunits are responsible for DNA sequence recognition and bonding, while the C subunit, which does not contact the DNA, protrudes out of the minor groove perpendicular to the duplex.45 The C subunit is not inert, it plays an important role in the cytotoxicity of Et743.46 It targets DNA by binding in the minor groove in G+C-rich sequences, alkylates the amino group of guanine residues at position 2,47 and bends DNA toward the major groove with an absolute curvature of 17(±3)°.45 The direction of bending is a novel

feature among minor groove DNA-interactive agents, making Et743 unique. Binding to the minor groove occurs through an extensive network of hydrogen bonds that presumably results in an increase in duplex stabilization by 13–19 deg. C.48 Following binding, alkylation of predominantly guanine bases, located either in the sequence 5′-PuGC or 5′-PyGC, occurs.48,49 Other adducts are formed, but these are less stable and are reversible.45,48 Et743 is active against soft tissue sarcomas, in breast and ovarian cancers and is currently in phase II/III clinical trials.50,51 It effectively disrupts transcription factor complexes in vitro and inhibits gene expression in vivo.52,53 Et743 DNA adducts also inhibit nucleotide excision repair enzymes both in vitro and in vivo during the excision step, causing the accumulation of DNA–drug–protein intermediates.36,54 Hedamycin, isolated from Streptomyces griseoruber, belongs to the highly cytotoxic pluramycin class of antibiotics (Figure 3(c)).55 It consists of a planar anthrapyrantrione chromophore attached to two amino sugar rings at one end and a bisepoxidecontaining side-chain at the other end (Figure 3(c)). Hedamycin binds to dsDNA by both reversible and non-reversible modes, and produces a 20 deg. C increase in the DNA melting temperature.56 Binding to DNA involves an initial reversible intercalation of the chromophore, followed by a much slower covalent binding to the DNA bases.57 Intercalation into DNA occurs in the major groove by a threading mechanism, and by interactions of the carbohydrate moieties with the major and minor grooves at 5′NGC sequences, which selectively aligns the epoxide in the major groove for the nucleophilic attack of the N7 of guanine. 58 Studies of the alkylated complexes of hedamycin and altromycin B (another pluramycin antibiotic) with oligonucleotide substrates demonstrate that the glycosidic substituents determine sequence selectivity.58–60 The modification of DNA by hedamycin results in inhibition of both DNA and RNA polymerases,56,61 and disruption of transcription factor–DNA complexes in vitro.62,63

DNA Alkylators Inhibit RecBCD Enzyme

To date, two studies evaluating the ability of anticancer agents to inhibit RecBCD enzyme have been done. Cisplatin and psoralen were evaluated for their ability to inhibit RecB and RecBCD, respectively. The anti-tumour activity of cisplatin is generally attributed to its formation of DNA adducts, both intrastrand and inter-strand crosslinks, which induce structural distortions in DNA. Cisplatin intra-strand DNA adducts inhibited both the DNA helicase and ATPase activities of RecB.64 In contrast, psoralen inter-strand cross-links inhibited the helicase activity of RecBCD, but had no effect on the hydrolysis of ATP, which is consistent with an uncoupling of the DNA helicase and ATPase activities of the enzyme.65 The goal of this study was to determine and characterize the effects of adozelesin, Et743 and hedamycin on the DNA helicase activity of the RecBCD enzyme. In contrast to previous work with other DNA helicases,8 and because the substrate for RecBCD is typically DNA molecules greater than several hundred base-pairs in length, we used plasmid-length DNA substrates (i.e. 4100 bp in length). Two complementary DNA helicase assays (fluorescence-based and agarose gel-based) were used to evaluate the effects of each drug on DNA unwinding. The results show that each drug is able to inhibit RecBCD to varying degrees, with Et743 being the most potent inhibitor. In addition to the helicase activity being inhibited, the agarose gelbased assay reveals that both the nuclease activity and the recognition and response to χ are also affected, with the effects being distinct for each agent. Analysis of the effects on the rate of ATP hydrolysis by the translocating RecBCD enzyme revealed that ATP hydrolysis is not uncoupled from DNA unwinding, although for Et743, a twofold decrease in the efficiency of ATP-utilization was observed. These results suggest that the druginduced structural changes to the DNA, combined with the increased melting temperature of the duplex, caused the rapidly translocating RecBCD enzyme to slow markedly and to push its way through the site(s) of modification. The ability of the enzyme to force its way through the site of damage provides the nuclease active site an increased opportunity to interact with the unwound ssDNA, resulting in increased nuclease activity. Finally, the results reveal a surprising inhibition of the binding of the E. coli ssDNA-binding protein (SSB) to drugmodified and unwound DNA, suggesting that this may be an additional means of disruption of DNA metabolic processes exerted by alkylating agents.

Results The fluorescence assay demonstrates that adozelesin, Et743 and hedamycin inhibit DNA unwinding by RecBCD To test whether the three alkylating agents could inhibit the helicase activity of RecBCD, a fluorescent

903 helicase assay was used initially.19 This assay takes advantage of the >106-fold higher affinity of the RecBCD enzyme for dsDNA ends relative to internal sites, 19 and monitors the unwinding of linear dsDNA by RecBCD in real-time using the intrinsic fluorescence of SSB as a reporter for dsDNA unwinding.19 In this assay, the RecBCD enzyme unwinds linear dsDNA, producing ssDNA, which is accessible to SSB. The SSB protein binds immediately to this ssDNA, resulting in quenching of the intrinsic protein fluorescence of SSB. The fluorescence decrease is monitored using a fluorescence spectrophotometer and reflects accurately the helicase activity of RecBCD as demonstrated previously.19 A typical DNA unwinding assay using unmodified χ°-dsDNA consists of three phases and is shown in Figure 4(a). In phase I, the fluorescence trace resulting from the intrinsic fluorescence of SSB is constant, as there is no ssDNA present. Phase II (indicated by the grey box) is initiated by the addition of RecBCD at t = 59 s (indicated by the arrow) and represents a rapid decrease in the

Figure 4. Adozelesin, Et743 and hedamycin inhibit DNA unwinding. (a) A time-course of DNA unwinding using stoichiometric conditions. Phases I–III are discussed in the text. The reaction was performed using standard conditions as described in Materials and Methods, and was initiated by the addition of RecBCD (indicated by the black arrow). The broken red line in phase II corresponds to the tangent line used to calculate the initial rate of DNA unwinding, during which the intrinsic fluorescence of SSB protein is quenched over time. The continuous red line in phase III indicates the extent of DNA unwinding or maximum level of SSB-fluorescence quenching (ΔF). (b) RecBCD DNA unwinding reactions using unmodified (control) and drug-treated dsDNA. The separate reactions contained DNA modified using an input concentration of 3 μM for each drug, corresponding to, on average, 99 drug molecules per DNA molecule.

904 fluorescence signal due to the quenching of the intrinsic fluorescence of SSB that occurs upon binding to the nascent unwound ssDNA produced by the helicase activity of RecBCD. In the reaction shown, unwinding is completed in approximately 60 s (at t = 120 s). In phase III, no change in the now reduced fluorescence signal is observed, indicating that all available dsDNA has been unwound (Figure 4(a)). The slope of the line (Δf/Δt) fitted to the data points in phase II is used to calculate the initial rate of unwinding, while the difference in total fluorescence (ΔF), represents the extent of unwinding (Figure 4(a)). The unwinding rate in this reaction was 99(±27) bp s−1 consistent with previous results,19,66,67 and the decrease of 40–50% of the intrinsic SSB fluorescence signal represents the extent of the reaction. The maximum achievable extent was established in separate, control reactions by adding heat-denatured linear dsDNA to reactions mixtures containing SSB alone. Here, a similar value of ΔF was observed (55– 65%; data not shown), indicating that under the conditions used, the majority of the DNA was unwound by RecBCD enzyme. The effects of each agent on RecBCD were evaluated separately in a single round of DNA unwinding (i.e. using stoichiometric conditions with one enzyme per DNA molecule). This was done to minimize the effects of unwound and modified DNA on subsequent unwinding of additional DNA molecules by an individual RecBCD heterotrimer. Initially, to determine whether each agent could affect the unwinding of dsDNA by RecBCD, the same concentration of each drug was used to alkylate dsDNA (3 μM), represented here as the number of drug molecules per DNA molecule. In these initial reactions, this corresponds to 99 drug molecules per DNA molecule, which is, on average, one drug molecule every 42 bp. After treatment with the drug, the DNA was used directly in the fluorescent helicase assay without further purification. The unwinding time-courses of control and drug-treated dsDNA unwinding reactions are presented in Figure 4(b). The data demonstrate that each agent inhibits both the rate and the extent of DNA unwinding. In contrast to the control reaction, the unwinding of adozelesin-modified dsDNA was biphasic. The first phase was reduced by 25% to 61(±28) bp s−1 and lasted approximately 20 s, while in the second phase, the rate was reduced fivefold relative to the control, to 18(±8) bp s−1, and lasted for an additional 160 s (Figure 4(b)). Even though treatment with adozelesin resulted in a reduction in the unwinding rate, RecBCD was able to unwind the DNA fully in 300 s. Alkylation of DNA by Et743 resulted in a 17fold reduction in the rate of unwinding by RecBCD from 99(±27) bp s−1 to 6 bp s−1. The extent of reaction was inhibited also with only 25% of the dsDNA being unwound by 360 s. By extrapolating the reaction curve, it is anticipated that Et743-modified DNA would be unwound completely but would require ∼25 min to occur (data not shown). Treatment of DNA with hedamycin resulted in the

DNA Alkylators Inhibit RecBCD Enzyme

unwinding rate being reduced 6.6-fold to 15 bp s−1 and the extent inhibited by 35%. By extrapolation of the fluorescence trace, RecBCD would completely unwind the modified DNA in approximately 8 min (data not shown). Thus, at the same concentration of the drug (i.e. the same drug to DNA molecular ratio), each alkylating agent inhibits RecBCD but different levels of inhibition are observed for each agent. The agarose gel assay provides additional insight to the mechanism of drug-induced inhibition of DNA unwinding The reduced rate and extent DNA unwinding observed in the fluorometric assay could be due to direct inhibition of translocation and DNA unwinding by RecBCD or to inhibition of SSB protein binding to modified and unwound ssDNA. As the fluorescence-based assay permits observation of the appearance of only unwound DNA (observed as a decrease in the intrinsic fluorescence of SSB), an agarose gel-based assay was used to provide complementary insight into the potential mechanism of RecBCD inhibition by each agent. In this assay (Figure 1(a)), both the helicase and nuclease activity of RecBCD on 5′-end labeled dsDNA can be observed as a function of time. The inhibitory effects of each drug on RecBCD during a single round of DNA unwinding (1 enzyme molecule per DNA molecule) were evaluated using experimental conditions similar to those used for the fluorometric assay (Figure 4). Under these conditions, using χ°DNA, RecBCD will simultaneously unwind the duplex and preferentially degrade the 3′-terminated strand relative to the entry point of the enzyme, while leaving the 5′-terminated strand essentially intact. The products of this reaction are full-length, ssDNA and a heterogeneous population of single-stranded nucleolytic fragments (Figures 1(a) and 5(a)). In the control reaction, DNA was unwound rapidly, at a rate of 103(±25) bp s−1, with complete unwinding occurring within 60 s. The rate and extent of unwinding by RecBCD were identical, within experimental error, with that observed in the fluorometric assay (103(±28) bp s−1 and 100%, respectively; Figure 4(a)). As expected, the disappearance of the dsDNA substrate was accompanied by the appearance of full-length ssDNA and a heterogeneous population of nucleolytic fragments (Figure 5(a), left panel). To permit direct comparison between the fluorometric and gel assays, the same concentration of each agent was used (i.e. 3 μM). After incubating with the individual drugs, drugmodified dsDNA was added to the reaction mix followed by RecBCD to initiate the reaction. At the indicated time-points, samples were removed, mixed with loading buffer, and subjected to agarose gel electrophoresis. The gel and resulting analysis are presented in Figure 5. In contrast to the fluorimetric assay, a reduction in the rate of DNA unwinding by RecBCD was

DNA Alkylators Inhibit RecBCD Enzyme

905

Figure 5. Alkylating agents affect both the helicase and nuclease activities of RecBCD. (a) Representative agarose gels of DNA-unwinding reactions are shown. The control and adozelesin are from the same gel, while the Et743 and hedamycin reactions are form separate gels. Linear dsDNA was treated with adozelesin, Et743 or hedamycin (87 drug molecules per DNA; 3 μM each), and subjected to strand separation by RecBCD under standard reaction conditions as described in Materials and Methods. At the foremost left side of the gel, the radioactively labeled dsDNA marker bands are indicated. (b) Analysis of the gels shown in (a). The amount of dsDNA present at each time-point is expressed as a percentage of the input amount of dsDNA for each reaction (zero time-point). The data represent the mean from at least two independent experiments (±SE, n ≥ 2).

observed only for adozelesin and Et743, and not for hedamycin. The inhibition by adozelesin observed here was modest, and the time-course was biphasic, similar to that observed in the fluorescence assay. There was an initial phase of unwinding in which only a small effect on rate was observed (74(±19) bp s−1), similar to that observed in the fluorescence assay (61(±28) bp s−1). This was followed by a longer, second phase in which the rate of unwinding was reduced 26-fold to 4(±3) bp s−1 (Figure 5(b)). The reduced unwinding rate induced by adozelesin was accompanied by the appearance of stable intermediates that migrated just below the dsDNA substrate (Figure 5(a), lanes 11–17). The positions of migration of these unwinding intermediates in the gel were consistent from experiment to experiment, and the level of these intermediate species became more pronounced at increased molecular ratios of adozelesin to DNA (≥2885 drug molecules per DNA, data not shown). For Et743, a 63% reduction in rate was observed, down from 103(±25) bp s−1 to 38(±13) bp s−1 (Figure 5(a) and (b)). Although the level of inhibition is not

as great as that seen in the fluorimetric assay, where >90% inhibition was observed, Et743 was still the most potent inhibitor of unwinding used here. The reduction in the rate of DNA unwinding was accompanied by an increase in the nuclease activity of the enzyme. This is observed as both a decrease in the amount of full-length ssDNA, and a decrease in the size of nucleolytic fragments (Figure 5(a)). In the control reaction, a heterogeneous population of nucleolytic fragments is produced, and this is visualized as a smear that migrates below the fulllength ssDNA (Figure 5(a), lanes 1–9). With Et743treated DNA, this heterogeneous smear is virtually undetectable, and is coincident with appearance of small degradation products migrating at the bottom of the gel (<0.8 kb, Figure 5(a), lanes 20–28). Surprisingly, and in sharp contrast to the fluorimetric assay, no effect of hedamycin-modification of dsDNA on the rate of unwinding was observed, resulting in a rate of 100(±8) bp s−1 (Figure 5(a) and (b)). As hedamycin treatment of dsDNA produced a significant inhibition of DNA unwinding in the fluorescence assay, this would

906 suggest that perhaps hedamycin does not inhibit unwinding by RecBCD, but instead adducted ssDNA may not be bound efficiently by SSB. It is not possible to assess the inhibition of SSB binding to hedamycin-modified ssDNA directly, as the heat-treatment required to produce ssDNA to assess binding results in breaks and terminal positioning of the adduct (for adozelesin and hedamycin) or loss of the adduct (Et743). Furthermore, and in contrast to the fluorimetric assay, where an 80% decrease in extent was observed with Et743-modified DNA (Figure 4(b)), no effect on extent was observed in the gel assay. As the level of nuclease activity is elevated on Et743modified DNA, this suggests that the nuclease fragments produced are either too small to be bound efficiently by SSB, or adducted-ssDNA is not bound efficiently by SSB. A similar disparity between the two assays was observed for hedamycin, although the results were not as dramatic. For each agent tested, the level of inhibition observed in the gel assay was not as high as that observed in the fluorescence-based assay. This difference could be due to drug-induced pausing of the enzyme close to the entry site (i.e. the dsDNA end) allowing the 5′-terminal strand to interact with the nuclease domain for an extended length of time. As the substrate used here is labeled at the 5′ end,

DNA Alkylators Inhibit RecBCD Enzyme

drug-induced pausing could result in label removal, thereby giving the appearance of DNA unwinding, and consequently an apparent reduction in the levels of inhibition relative to the fluorescencebased assay. To determine whether DNA unwinding was in fact occurring, DNA unwinding assays were conducted exactly as in Figure 5(a) using drug-treated and radioactively labeled DNA. Following electrophoresis, gels were stained with SYBR-green and photographed. The same gels were subsequently dried, exposed to Phosphorimager screens and analyzed. The results show that there is essentially little or no difference in the rate and extent of unwinding when using either SYBR-green or radioactive labeling to monitor the amount of substrate remaining (Figure 6). However, at a tenfold higher concentration of Et743, where ∼90% inhibition of the rate of unwinding is observed, a difference between the two methods of substrate detection is observed (Figure 6(c)). This suggests that at extremely high drug to DNA molecular ratios, DNA alkylation may induce RecBCD to stall close to the entry site where it removes the 5′-end label. In the gel assay, this would give the appearance of DNA unwinding. Therefore, for the majority of concentrations of drug tested (including those in the following section), the radioactive gels demonstrate DNA unwinding and

Figure 6. The inhibition of RecBCD by DNA alkylation is not simply dsDNA end-induced stalling resulting in removal of the radioactive label. (a) Control unwinding reactions using unmodified DNA. (b)–(d) Drug-treated DNA. Radioactively labeled dsDNA (285 μM in nucleotides) was treated with (b) 2884 adozelesin molecules per DNA molecule; (c) 87 or 865 Et743 molecules per DNA molecule, squares and circles, respectively; (d) 865 molecules of hedamycin per DNA molecule. For hedamycin treatment only, DNA was precipitated to remove free hedamycin before being used in DNA helicase assays. Following electrophoresis, gels were stained with SYBR green for 30 min and then photographed. The same gels were dried and exposed to Phosphorimager screens. Open symbols, quantification using radioactivity. Filled symbols, quantification using SYBR-green staining. The amount of DNA remaining at each time-point is expressed as a percentage of the input dsDNA substrate.

907

DNA Alkylators Inhibit RecBCD Enzyme

not simply removal of the radioactive label by RecBCD paused close to the dsDNA entry point of the enzyme. Furthermore, each agent does reduce the rate of unwinding relative to the control reaction, but clearly, the level of inhibition is not as severe as that observed in the fluorescence-based assay. The different levels of inhibition are not due to differences in affinity of adozelesin and hedamycin for dsDNA The different levels of inhibition observed for each drug could be attributed to differences in the affinity of each agent for the DNA as well as to the levels of free, unbound drug molecules. These unbound molecules could conceivably bind directly to RecBCD or SSB and inhibit their activity or binding, respectively. Since each compound binds dsDNA and rapidly undergoes covalent attachment, it is not possible to measure binding constants easily. Consequently, the level of DNA damage induced by the binding of each agent to DNA and the amount of free drug was determined using a modified version of a heat-induced, DNA strand cleavage assay, as described in Materials and Methods.68 This assay uses heat to induce DNA strand cleavage at the site of DNA alkylation. Here, we used two differentsized supercoiled DNA plasmids as substrates and assessed the loss of supercoiled DNA following heating, by quantification of agarose gels used to separate the different forms of DNA. The level of superhelical plasmid loss (i.e. DNA strand cleavage) corresponds to the amount of drug bound to the DNA at the time of heating. Cleavage of the first plasmid is used to assess initial binding of an agent to DNA, while cleavage of the second plasmid is used to assess the level of free drug molecules present in solution, following the original modification. As heating reverses the covalent binding of Et743 to DNA,47,48 this agent could not be tested and only the damage induced by adozelesin and hedamycin could be assessed. Therefore, we reasoned that if adozelesin and hedamycin bind to dsDNA with similar affinities, then the level of induced damage observed following heat treatment should be the same. Furthermore, if free drug is present after the first incubation, then it would bind to the second plasmid and DNA damage would be observed in this plasmid as well. The results demonstrate that at 0.3 μM adozelesin or hedamycin (nine drug molecules per DNA molecule), DNA modification resulted in the loss of 45% and 26% of the first supercoiled plasmid, respectively (data not shown). Further, for adozelesin, 45% of plasmid 2 was cleaved, while 39% was cleaved following treatment with hedamycin (data not shown). Thus, the affinity of adozelesin for DNA is higher than that of hedamycin (by approximately twofold), and the amount of free drug remaining is comparable. Thus, the different levels of inhibition observed are due, in part, to the differences in affinity of adozelesin and hedamycin for DNA, and

to the presence of free drug molecules in the reaction. The effects of free drug molecules on RecBCD are addressed below. Free hedamycin inhibits the helicase activity of RecBCD In addition to the effects of the alkylating agents on SSB binding to modified and unwound single strands of DNA, the presence of free, unreacted drug molecules could contribute to the inhibition of DNA unwinding by RecBCD. Conceivably, these could bind to RecBCD directly, thereby altering the activity of the enzyme. To determine whether this was occurring, reactions using drug-treated, ethanol-precipitated dsDNA were evaluated. Precipitation in ethanol has been demonstrated to remove free drug molecules and/or terminate the alkylation reaction.43,69 Therefore, if inhibition of unwinding is observed using drug-modified and ethanol-precipitated dsDNA, the inhibition can be attributed to DNA-bound drug molecules. Drug to DNA molecular ratios that inhibited the unwinding rate of RecBCD by >50% as determined in the agarose gel assay were used, corresponding to 2884 adozelesin, 288 Et743 and 865 hedamycin molecules per DNA molecule, respectively. Following treatment with the drug, 5′-end labeled DNA was precipitated in ethanol, resuspended in water and used directly in the agarose gel assay. The results are summarized in Table 1. The level of inhibition observed using both adozelesin and Et743-treated DNA was the same using either nonprecipitated or precipitated dsDNA (Table 1). Thus, inhibition of unwinding by adozelesin and Et743 is attributed to DNA-bound drug molecules. Even though the heat-induced, DNA strand cleavage assay demonstrated the presence of free adozelesin molecules, these free molecules do not appear to affect the helicase activity of RecBCD adversely. In contrast, a significant difference in the rate of unwinding between hedamycin-modified, precipitated and non-precipitated DNA was observed. The rate of unwinding increased 1.6-fold from 37(±9) bp s−1 to 63(±11) bp s−1 when the DNA was precipitated Table 1. Free hedamycin affects the unwinding of dsDNA by RecBCD

Control Adozelesin Et743 Hedamycin a

Non-precipitated dsDNA

Precipitated dsDNA

Drug/ dsDNAa

DNA unwinding rate (bp s−1)b

DNA unwinding rate (bp s−1)b

− 2884 288 865

94 ± 15 39 ± 11 28 ± 8 37 ± 9

91 ± 11 40 ± 22 31 ± 18 63 ± 10

The drug to DNA molecular ratios correspond to the following concentrations: 100 μM adozelesin, 10 μM Et743 and 30 μM hedamycin. b RecBCD rate of DNA unwinding expressed as base-pairs (nM) s−1 RecBCD enzyme nM−1, was established on the basis of the disappearance of the radioactive substrate in the agarose gel assay under standard reaction conditions.

908 in ethanol (Table 1). The rate of unwinding of hedamycin-treated and ethanol-precipitated DNA was still reduced by 35% relative to the control unmodified DNA, indicating that DNA-bound hedamycin does inhibit the progress of the translocating RecBCD enzyme (Table 1). Therefore, to minimize the effects of free hedamycin on RecBCD, subsequent experiments used hedamycin-treated and ethanol-precipitated dsDNA. Adozelesin, Et743 and hedamycin inhibit the rate of dsDNA unwinding by RecBCD and not the extent of reaction To further characterize the drug-induced inhibition of DNA unwinding, a titration of each agent relative to DNA was done, and the effects were assessed using both the fluorescence-based and gelbased helicase assays. This is expected to reveal the drug concentrations that inhibit enzyme activity by 50% compared to the control (IC50). To permit direct comparison, we present data as a function of the ratio of drug molecules per DNA molecule rather than as the total drug concentration present in the alkylation reaction. Drug concentrations ranging from 3 to 865 drug molecules per DNA molecule were used in separate reactions to modify dsDNA, and the effects of each drug on the rate and extent of unwinding by RecBCD were determined. The resulting rates and extents of reaction presented in Figure 7 are expressed as a percentage of the control rates and extents of reaction, respectively, as observed with unmodified dsDNA. As expected, the results show that, although each agent inhibits the helicase activity of RecBCD, the IC50 for the rate and extent of unwinding occurred at different drug to DNA molecular ratios for each agent. Adozelesin inhibited the rate of DNA unwinding in the fluorescence assay by 40% at 100 drug molecules per DNA molecule (Figure 7(a)). No additional decrease was observed up to ∼330 drug molecules per DNA molecule, while almost complete inhibition of unwinding was observed at higher concentrations. For this agent, the IC50 is ∼350 drug molecules per DNA molecule (i.e. one drug molecule, on average, every 12 bp). In contrast, for Et743, the IC50 was observed at ten drug molecules per DNA molecule (or, ∼1 every 400 bp) with total inhibition being observed at 30 drug molecules per DNA molecule (Figure 7(b)). Hedamycin demonstrated an IC50 similar to that of Et743 (∼20 drug molecules per DNA molecule), although in contrast to Et743 and adozelesin, complete inhibition of the rate of unwinding was not observed (Figure 7(c)). In contrast to the effects on the rate of DNA unwinding by RecBCD, the effects on the extent of reaction were quite different. The IC50 for the extent of reaction was observed at 440 molecules of adozelesin and 99 molecules of Et743 per DNA molecule (Figure 7(a) and (b), respectively). At these concentrations, the rate of unwinding was reduced to 30% and 10% of the control, respectively. For hedamycin, 50% inhibition could not be achieved

DNA Alkylators Inhibit RecBCD Enzyme

even at the highest concentration used (350 drug molecules per DNA molecule), while the rate of unwinding was reduced to 25% compared to the control (Figure 7(c)). To separate the effects of alkylating agents on the helicase activity of RecBCD from that on SSB binding, a titration of each agent was done using the agarose gel assay. The rates and extents of unwinding were determined as described for Figure 5 and are expressed as a percentage of the control reaction and graphed as a function of the drug to DNA molecular ratio (Figure 7(d)–(f)). As before, each alkylating agent inhibited RecBCD at different drug to DNA molecular ratios and, consistent with the fluorescence data, Et743 was the most effective inhibitor. However, several differences between the IC50 values obtained in each assay exist. First, the IC50 is significantly higher in the gel assay than in the fluorescence-based assay. For adozelesin it is 2.5fold higher, for Et743 it is sixfold higher, and for hedamycin it is 54-fold higher. Second, and in contrast to the fluorescence assay, adozelesin and hedamycin were found to be equally effective in the gel assay, with a 50% inhibition of the rate of unwinding being observed at 430 drug molecules per DNA molecule (Figure 7(d) and (f)). Third, treatment with adozelesin and Et743 had little or no effect on the reaction extent, while hedamycin was effective but only at extremely high concentrations (>3000 molecules per DNA molecule, Figure 7(f)). At these elevated concentrations of hedamycin, it may be difficult to remove all of the unbound hedamycin, so it is not possible to separate the effect of free versus DNA-bound drug molecules on the translocating RecBCD enzyme. In summary, the results from these assays demonstrate that each agent inhibits the rate of unwinding of RecBCD, but to varying degrees. Et743 was the most potent inhibitor of DNA unwinding, followed by adozelesin and hedamycin. As the gel assay permits direct observation of the unwinding of dsDNA and is not simply removal of the 5′-end label, and the fluorescence assay requires SSB binding to ssDNA to detect unwinding, the enhanced level of inhibition seen in the fluorescence assay is due to both inhibition of RecBCD unwinding and, likely, binding of SSB to drug-modified and unwound DNA. Under catalytic conditions, modification by adozelesin traps the translocating RecBCD enzyme All the experiments described thus far were done using stoichiometric conditions, that is, one RecBCD molecule per DNA molecule. To determine whether alkylating agents could inhibit RecBCD during subsequent rounds of DNA unwinding, RecBCD activity was evaluated under catalytic conditions, that is, one RecBCD molecule per four DNA molecules. Using a low concentration of the drug (29 drug molecules per DNA molecule), the effects of each agent on the catalytic behavior of RecBCD

DNA Alkylators Inhibit RecBCD Enzyme

909

Figure 7. The fluorescence and gel-based assays demonstrate that alkylating agents inhibit the helicase activity of RecBCD in a concentration-dependent manner. The effects of increasing concentrations of alkylators on the rates and extents of DNA unwinding are shown. (a)–(c) Fluorescent-based assay; (d)–(f) agarose gel assay. For (a)–(c), the dsDNA (250 μM in nucleotides) was incubated with increasing concentrations of drug varying from 33 to 986 molecules per DNA molecule for adozelesin, and from 3 to 99 drug molecules per DNA molecule for Et743 and hedamycin. The rates and extents derived from DNA-unwinding reactions are presented together on the same graph as a function of (a) adozelesin, (b) Et743 or (c) hedamycin molecules per DNA molecule. Data points represent the values derived from at least two independent experiments ± standard error. For (d)–(f), DNA was treated with concentrations of drug ranging from (d) 87 to 865 adozelesin molecules per DNA molecule, (e) from 9 to 288 Et743 molecules per DNA molecule, and (f) from 87 to 28,835 hedamycin molecules per DNA molecule. The rate of unwinding was determined by fitting a straight line to the early data points of the time-courses, such as those shown in Figure 4(c). The resulting rate is expressed as a function of the rate of unwinding of unmodified DNA (103 ± 25 bp s−1). The extent of unwinding was determined from the intensity of dsDNA remaining 20 min after incubation with RecBCD. Plotted points indicate data from multiple experiments (±SE, n ≥ 2).

were determined using the agarose gel assay. The rates were determined as described previously and the results are presented in Table 2. The rate of unwinding of unmodified dsDNA was as expected, within experimental error, the same using both catalytic and stoichiometric con-

ditions (98 and 103 bp s−1, respectively, Table 2). The results using drug-treated dsDNA were the same under both catalytic and stoichiometric conditions for Et743 and hedamycin. Thus, treatment of the DNA with these two agents does not appear to alter the RecBCD enzyme, nor does it

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DNA Alkylators Inhibit RecBCD Enzyme

Table 2. The rates of DNA unwinding under stoichiometric and catalytic conditions are the same

Druga Control Adozelesin Et743 Hedamycin

DNA unwinding rate (bp s−1)b stoichiometric reaction

DNA unwinding rate (bp s−1)b catalytic reaction

103 ± 29 70 ± 17 – 15 ± 3 95 ± 34

98 ± 7 84 ± 10 17 ± 3 17 ± 2 75 ± 8

a The same concentration of each agent was used, that is 1 μM or 29 drug molecules/DNA molecule. b RecBCD rate of DNA unwinding expressed as base-pairs (nM) s−1 RecBCD enzyme nM−1, was established on the basis of the disappearance of the radioactive substrate in the agarose gelbased assay under standard conditions, as described in Materials and Methods.

result in enzyme entrapment during translocation and DNA unwinding. In contrast, adozelesin induced two distinct rates of DNA unwinding: a rapid rate of 84 bp s−1, lasting for 30 s, followed by a second phase lasting more than 5 min (17 bp s−1, Table 2). Similar results were obtained using stoichiometric conditions in both the fluorescence-based and gel-based assays (Figures 4(c) and 5). The two phases of unwinding are consistent with transient, adozelesin-induced enzyme entrapment or stalling occurring during the initial DNA unwinding, resulting in the formation of stable intermediates in agarose gels (Figure 5). The inhibition of RecBCD occurs only during the unwinding of dsDNA and is not a visible, permanent alteration of the enzyme. This is supported by the comparison of initial rates of DNA unwinding under both stoichiometric and catalytic conditions (Table 2). To further verify that drug-induced modification does not alter the enzyme permanently, fluorescence helicases assay were carried out using three different concentrations of drug for each agent (0.3 μM, 3 μM and 30 μM). Once the reactions had proceeded to completion, as indicated by no additional change in the fluorescence signal, a second aliquot of unmodified DNA was added. This was unwound at the same rate and to a similar extent as that observed in the control reaction using unmodified DNA (data not shown). Thus, the progress of the translocating RecBCD enzyme is inhibited during progression through drug-modified DNA but is not altered permanently by its interaction with adducted DNA. Alkylating agents alter the ability of RecBCD to recognize and respond to the recombination hotspot χ The translocating RecBCD enzyme recognizes and is regulated by the cis-acting recombination hotspot χ. This 8 nt sequence is recognized as the single strand, unwound form only when the enzyme approaches it from the 3′-side.25 Upon encountering χ, several changes in the translocating enzyme

occur. First, the enzyme pauses,26 then the polarity of DNA degradation is altered: cleavage of the 3′terminated relative to the entry site of the enzyme is attenuated, while degradation of the 5′-terminated strand is up-regulated.27 Continued unwinding past χ results in preservation of the χ-containing strand that is used in homologous recombination. Using 5′end-labeled dsDNA containing an asymmetrically positioned χ sequence, the recognition of, and response to χ by RecBCD can be visualized readily using the agarose gel assay (see the schematic in Figure 1(b)). If RecBCD enters from the left of the substrate as shown, χ is in the incorrect orientation and consequently will not be recognized. If RecBCD enters the substrate from the right, χ will be recognized and, consequently, the resulting top strand, downstream and bottom strand, upstream χ-specific fragments will be generated (Figure 1(b)). To test whether alkylating agents alter the ability of RecBCD to recognize and respond to χ during a single round of DNA unwinding, χ-containing, 5′end-labeled DNA was used. As the efficiency of χ recognition is only 30%, we used a tandem χ array containing three χ sequences, with 10 bp of intervening sequence. This array ensures 100% recognition efficiency.26 In this work, we utilized the same plasmid DNA as χ° and χ+, in attempts to minimize sequence-specific effects of drug-modification between different plasmids. Thus, the same plasmid DNA was used as in the previous experiments (i.e. pPB12 linearized with EcoRI), except here it was linearized with BamHI. Restriction with EcoRI positions the χ array within 50 bp of the entry point of the enzyme, so that it is not recognized efficiently (data not shown). In contrast, cleavage with BamHI positions the χ array 1100 bp near one end of the dsDNA substrate. Thus, upon χ recognition, RecBCD would produce two unique ssDNA fragments: a 3000 nt fragment, designated as the upstream, bottom strand χ-specific fragment and an 1100 nt fragment designated as the downstream, top-strand χ-specific fragment. To allow RecBCD to translocate the full length of the dsDNA to ensure the possibility of χ being recognized, concentrations of drug that decreased the rate of unwinding of χ°-dsDNA by less than 50% were used. These corresponded to a drug to DNA molecular ratio of 87 for adozelesin, 29 for Et743 and 865 for hedamycin. Following treatment with the drug (and precipitation in ethanol for hedamycin-treated DNA), unwinding reactions were initiated with RecBCD and reaction products were resolved from substrate and intermediates by agarose gel electrophoresis. Control reactions using untreated dsDNA were done in parallel and subjected to electrophoresis in the same gels to permit direct comparison (Figure 8, lanes 1–9). Unwinding of control dsDNA occurred at a rate of 120 bp s−1 and was completed in 30 s, similar to that observed for untreated χ°-dsDNA (Figure 5). Concomitant with dsDNA unwinding, full-length ssDNA and the expected 3000 nt upstream, bottom-strand, χ-specific fragment (Figure 8,

DNA Alkylators Inhibit RecBCD Enzyme

911

Figure 8. DNA alkylating agents alter the ability of RecBCD to recognize and respond to χ. A representative gel showing χ-fragment production by RecBCD using unmodified, adozelesin, Et743 or hedamycin-treated dsDNA. The DNA substrate was produced by cleaving pPB12 with BamHI to position χapproximately 1100 bp from one end, as shown in Figure 1(b). The concentrations of each agent used to modify DNA are 3 μM adozelesin (87 drug molecules per DNA molecule), 1 μM Et743 (29 drug molecules per DNA molecule), and 30 μM hedamycin (865 drug molecules per DNA molecule). Hedamycin-treated DNA was precipitated in ethanol to remove free drug. Arrows (i) and (ii) indicate the position of bottom and top, χ-specific fragments, respectively. Adozelesin-treated DNA resulted in the accumulation of DNA unwinding intermediates (iii) and uniquely sized, apparent χ-specific fragments (iv) and (v), indicated by arrows.

arrow (i)) and the 1100 nt downstream, topstrand χ-specific fragment (Figure 8, arrow (ii)) were observed, indicating that the translocating RecBCD enzyme recognized and responded to χ (Figure 8, lanes 1–9). In contrast, the results with drug-treated dsDNA were quite different. First, as expected, treatment with adozelesin, Et743 and hedamycin decreased the rate of unwinding to 88 bp s−1, 68 bp s−1and 58 bp s−1 respectively, consistent with the rates observed using χ°-DNA (Figure 5). Second, drug modification of the dsDNA resulted in an alteration of the ability of the translocating RecBCD enzyme to recognize and respond to χ. This is visualized as a change in both the level and pattern of χ-specific fragment production, as discussed in detail below. In reactions containing adozelesin-treated dsDNA, several unique fragments were formed (Figure 8, lanes 10–18). First, discrete-sized and stable unwinding intermediates were formed that migrated between the substrate and full-length ssDNA (indicated by region (iii)). The position of migration of these intermediates is different from those observed during the unwinding of χ°-DNA (compare Figure 5(a), lanes 10–18 with Figure 8, lanes 10–18). Second, although the DNA substrate was unwound completely, formation of the anticipated χ-specific fragments did not occur. Instead, “apparent” χ-specific fragments were produced by RecBCD that migrated between the χ-specific fragments produced in the control reaction and well below the 1100 nt fragment (Figure 8, arrows (iv) and (v), respectively). As expected for Et743 and as observed for χ°DNA, treatment with the drug resulted in a decreased rate of unwinding and increased nuclease activity of the enzyme (Figure 8, lanes 21–29). As for χ°-DNA, a heterogeneous population of nucleolytic

fragments <800 bp in size was produced, consistent with elevated nuclease activity of the enzyme. Furthermore, the ability of RecBCD to recognize and respond to χ embedded within dsDNA is abolished by Et743-induced modification of the DNA substrate. A fragment is produced that migrates ahead of the position of the anticipated top-strand, downstream, χ-specific fragment. However, the production of this fragment is less than expected and, further, the appearance of this fragment is not coincident with DNA unwinding. Likely, this fragment arises due to post-unwinding degradation by RecBCD (since SSB binding to Et743modified DNA may not occur) and is not due to efficient recognition of χ. Similar to χ°, hedamycin-treated χ+-DNA was unwound with a decreased rate (58 bp s−1) and the amount of full-length ssDNA was reduced by 80% relative to the control (Figure 8). In contrast to the activity of the enzyme on adozelesin-modified and Et743-modified DNA, the translocating RecBCD enzyme is able to recognize and respond to a χsequence embedded within hedamycin-treated dsDNA. However, only the 1100 nt, top-strand, downstream χ-specific fragment was generated. The bottom-strand, upstream χ-specific fragment was not detectable and simultaneously, the level of full-length ssDNA was reduced significantly (Figure 8, lanes 30–38). As the enzyme is still able to recognize and respond to χ, the failure to produce the 3000 nt fragment is consistent with constitutive activation of the 5′-nuclease activity of the enzyme. Thus, modification of DNA by each agent results in unique effects on the ability of RecBCD to recognize and respond to χ. For Et743, this activity is inhibited completely, for adozelesin “apparent” χ-specific fragments are formed, while for hedamycin, χ-recognition still occurs but the response is

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DNA Alkylators Inhibit RecBCD Enzyme

aberrant due to the constitutive activation of the bottom-strand nuclease activity. DNA alkylators do not uncouple ATP hydrolysis from DNA unwinding The unwinding of dsDNA by RecBCD is coupled to the hydrolysis of ATP, with two to three ATP molecules being hydrolyzed on average, for each base-pair unwound.19 DNA unwinding and ATP hydrolysis of RecBCD can be uncoupled using psoralen, an inter-strand cross-linking agent.65 To test whether these intra-strand alkylating agents could inhibit the ATPase activity of RecBCD, ATP hydrolysis was measured using a spectrophotometric assay as described.70 The drug to DNA molecular ratios of each agent used (adozelesin, 865; Et743, 87; hedamycin, 2884 molecules per DNA molecule, respectively), resulted in a 60–90% inhibition of the helicase activity of the enzyme, as determined using the fluorescence-based DNA helicase assay (Figure 6 and Table 3). The rate of ATP hydrolysis for RecBCD using unmodified DNA was 313 s−1 (Table 3). The rate of DNA unwinding determined under identical conditions was 103 s−1. Using these rates, the ATP utilization of the enzyme per base-pair unwound can be calculated. For unmodified DNA, three ATP molecules are hydrolyzed per base-pair unwound, consistent with previous results.70 For adozelesinadducted dsDNA a 2.4-fold reduction in the rate of ATP hydrolysis was observed, while for Et743 and hedamycin, the rate decreased by 2.7-fold and 4.4fold, to 118 s−1and 80 s−1, respectively (Table 3). The rate of unwinding determined under identical conditions using drug-modified DNA was reduced twofold for adozelesin, 5.4-fold for Et743 and 4.8fold for hedamycin, as expected. Consequently, using these rates for DNA unwinding and ATP hydrolysis, the number of ATP molecules hydrolyzed per base-pair unwound for adozelesin and hedamycin, was calculated to be 2.5 and 3.5, Table 3. Intra-strand alkylating agents do not significantly perturb the allosteric interaction that activates the enzyme for ATP hydrolysis

Drug Control Adozelesin Et743 Hedamycin

Drug/ dsDNAa − 865 87 2884

kcat for DNA kcat for ATP ATP unwinding hydrolysis utilization (s−1)b (s−1)c (mol bp−1)d 103 ± 18 51 ± 22 19 ± 1 23 ± 9

313 ± 99 128 ± 5 118 ± 47 80 ± 13

3.0 2.5 6.2 3.5

a The drug to DNA molecular ratios correspond to the following drug concentrations: 30 μM adozelesin, 3 μM Et743 and 100 μM hedamycin. b RecBCD average rates established on the basis of the gel assay. c ATPase activity was measured using the spectrophotometric assay described in Materials and Methods. d The efficiency of ATP utilization per base-pair was derived from the maximal values of ATP hydrolysis (μM s−1) compared to the initial rate of DNA unwinding and calculated as described.19

respectively. In contrast, only a slight decrease in ATP utilization efficiency was observed for Et743 (6.2 ATP molecules per base-pair unwound; Table 3). Thus, the reduced rate of unwinding parallels and is coupled to a similar reduction in the rate of ATP hydrolysis. Therefore, these agents do not uncouple ATP hydrolysis from DNA unwinding.

Discussion The primary conclusion of this paper is that intrastrand alkylating agents inhibit the helicase and ATPase activities of RecBCD enzyme, but these do not become uncoupled. Further, the results demonstrate that each agent affects the nuclease, helicase and ATPase activities of RecBCD, but to varying degrees, and that the effects are complex and unique to each agent. Et743 was the most potent inhibitor of the helicase activity of RecBCD. Its modification of dsDNA resulted in a complete inhibition of the ability of the translocating enzyme to recognize and respond to χ. Adozelesin inhibited the progress of the translocating RecBCD enzyme through the DNA, resulting in transient trapping of the enzyme. In addition, modification by adozelesin resulted in the production of “apparent” χ-specific fragments, resulting from “pseudo-χ” recognition, as explained below. Hedamycin was the least potent inhibitor of unwinding, but its modification of dsDNA resulted in constitutive activation of the bottom-strand nuclease activity of RecBCD enzyme while leaving the ability of the enzyme to recognize and respond to χ intact. Two of the agents used, adozelesin and Et743, bind to dsDNA in the minor groove and were the most potent inhibitors of RecBCD, demonstrating that interactions with the minor groove play a critical function during translocation and DNA unwinding by this enzyme. Residues 245 through 254 of the leading domain of RecB are intimately associated with the DNA duplex in the minor groove. As this domain is responsible for pulling the DNA duplex into the holoenzyme (Figure 2(b)),31 disruption of these contacts delays the progress of dsDNA into the holoenzyme. Et743 was the most potent inhibitor of RecBCD activity used and its interaction with the DNA results in a significant distortion of the DNA structure, so that the minor groove is widened and the DNA is bent by 17(±3)° towards the major groove.45 Thus, one aspect of the mechanism of inhibition by Et743 is to disrupt the interaction of the protein with duplex DNA ahead of the translocating enzyme and thereby delay the entry of dsDNA into the enzyme. Additional inhibition by Et743 arises due to the increase in duplex stability by 19 deg. C.48 This stabilization may hinder the ability of RecBCD to split the duplex, further delaying the progress of the DNA into and/or through the enzyme. Finally, intra-strand alkylation by Et743 results in the covalent attachment of an adduct that remains covalently bound to the unwound ssDNA and requires heating to be removed.47,48 This adduct is

DNA Alkylators Inhibit RecBCD Enzyme

14–16 Å in size and is as large as the diameter of portions of the channels in RecC that the unwound DNA passes through (11–16 Å in diameter; Figure 2(b)). Thus, the progress of the Et743-adducted, unwound ssDNA through RecC is impeded, resulting in an overall reduction in the rate of DNA translocation. The above-mentioned effects combine to slow the passage of duplex DNA into, and ssDNA through the enzyme, thereby providing the nuclease domain additional time to interact with the unwound ssDNA, resulting in more frequent cleavage of the unwound ssDNA. This is visualized as an increase in the level of a subpopulation of short ssDNA fragments migrating at the lower parts of the gel, and a decrease in the level of full-length unwound ssDNA (Figures 5 and 8). Even though Et743 modification of DNA delayed the progress of the translocating enzyme significantly, it did not trap RecBCD on DNA, nor did it alter the activity of the enzyme significantly. Under catalytic conditions, no loss of enzyme activity was observed (Table 2), nor were stable unwinding intermediates observed in agarose gels (Figures 5 and 7). To further verify that the enzyme was not being altered permanently, unmodified dsDNA was added to unwinding reactions using Et743-modifed DNA that had been allowed to proceed to completion. This second aliquot of DNA was unwound at a rate comparable to that of control reactions (data not shown). This indicates that the modification of DNA by Et743 affects the translocating RecBCD enzyme while it is in transit through the DNA. Enzyme trapping or inactivation by the drug was observed, however, at high concentrations of Et743 (288 drug molecules per DNA molecule; data not shown). This drug to DNA molecular ratio corresponds to an average of one drug molecule every 14 bp, a value within the limits of the translocation step size of the enzyme of 23 nt.71 Presumably, at these high concentrations of Et743, each time the enzyme attempts to translocate 23 bp on dsDNA, it could do so into a region of modification. Since the modification by Et743 disrupts the interaction of the leading domain of RecB with the DNA, this would prevent the enzyme from moving, resulting in trapping or sequestering of the enzyme. Even though RecBCD is able to unwind and cleave Et743-modified dsDNA, χ-recognition was completely inhibited. This occurs due to the sequence selectivity of Et743 (5′-AG*C, 5′-GG*C) which overlaps that of χ at both the 5′-end and the 3′-end (5′-GCTGGTGG-3′). The recombination hotspot χ is recognized as the unwound single strand of DNA by the RecC subunit as the 3′-terminated DNA strand passes through the channel in RecC.15,25 The addition of the bulky adduct to χ impairs the ability of the RecC subunit to read the sequence, thereby preventing the recognition and response to χ from taking place. Modification of the χ sequence impairing the ability of RecBCD to recognize χ is not without precedence. A similar impairment has been observed with alternate χ sites, mutated at the 5′-G of the octanucleotide

913 sequence, resulting in a reduction in χ-dependent recombination proficiency to 6% relative to that of wild-type χ.72 Thus, the proposed modification of χ by Et743 results in a similar inability of RecBCD to recognize the unwound, single-stranded χsequence, resulting in elimination of χ-fragment production. The ability of Et743 to affect accurate sequence reading of unwound DNA by RecBCD may be applicable to enzymes such as RNA polymerase, which reads the sequence of the template strand during transcription. Here, the presence of bulky adducts could both delay the progress of the advancing transcription machinery, and alter its ability to read the sequence accurately and to transcribe the DNA accurately. Adozelesin was able to inhibit RecBCD but clearly was not as effective as Et743. The result of adozelesin-induced modification of dsDNA is to bend, stiffen and stabilize the duplex by 20 deg.C.39 The combination of these effects results in biphasic timecourses in both helicase assays, and the production of discrete-sized intermediates and apparent χspecific fragments in agarose gels. These effects are linked and can be explained by two models. In the first model, modification by adozelesin results in trapping of RecBCD during translocation and DNA unwinding. In the fluorimetric assay, using stoichiometric amounts of enzyme and 99 adozelesin molecules per DNA molecule, a brief (3 s) initial phase of unwinding was observed, followed by a second, longer phase in which a slower rate of unwinding was observed (18(±8) bp s −1 ). The biphasic time-course suggests that RecBCD is able to unwind a small segment of the duplex (approximately 363 bp) with a reduced rate (61(±28) bp s−1) relative to that observed on unmodified dsDNA (105(±27) bp s−1). During this period, eight to nine adozelesin molecules or, on average, one every 42 bp are encountered, resulting in the progress of the enzyme being impeded. In addition, and during this period, some fraction of the DNA-bound enzyme population becomes trapped or sequestered on the DNA. This results in the formation of unwinding intermediates that are visualized readily in agarose gels (Figures 5(a) and 8). In the gel shown in Figure 8, at early time-points, 14% of the input dsDNA is converted to these intermediate species. These persist in the gel to later times, with approximately 50% of these being unwound. This unwinding occurs at a considerably slower rate and is due to that fraction of the population of enzymes that completely unwound their respective DNA molecules, or possibly dissociated prematurely from their substrate molecules. These enzyme molecules unwind the intermediates by entering the substrate from the end opposite to which a sequestered enzyme is positioned. The encounter then displaces the previously trapped enzyme molecule, which does not appear to have been altered. To demonstrate that the enzyme was not being altered permanently, unmodified dsDNA was added to unwinding reactions using adozelesin-modified DNA that had been

914 allowed to proceed to completion. This second sample of DNA was unwound at a rate comparable to that of control reactions (data not shown). This indicates that the modification of DNA by adozelesin sequesters but does not permanently affect the RecBCD enzyme. In the second model to explain the biphasic timecourses, modification by adozelesin results in a modification of the RecBCD enzyme similar to that induced by the recombination hotspot χ. During translocation and unwinding of χ-containing DNA at 37 °C, RecBCD unwinds at 900 bp s−1 before χ. It pauses at χ for 5(±0.5) s, becomes modified, and the resulting unwinding rate following the pause is reduced to 143 bp/s−1.26 It was proposed that before χ, both the RecB and D motors are active. During the pause, RecD is inactivated by an unknown mechanism and the resulting reduced rate following χrecognition is presumed to be due to translocation by the RecB motor. Here, the sites of modification by adozelesin may induce the translocating RecBCD enzyme to pause, as it would when encountering a correctly oriented and positioned χ-sequence. During this pause, the RecD subunit becomes inactivated. When translocation and unwinding resume, the altered complex, which has only one motor operating, translocates more slowly. This would result in biphasic time-courses. The 41% reduction in unwinding rate to 61(±28) bp s−1 in the early phase of these time-courses suggests that dual motors in RecBCD may be able to unwind a limited amount of adozelesin-modified DNA, whereas the single motor complex with only RecB active has great difficulty in unwinding adozelesin-modified DNA, as indicated by the 82% reduction in unwinding rate to 18(±8) bp s −1 . If pseudo χ modification of RecBCD by adozelesin is occurring, then apparent χ-specific fragments should be visible in agarose gels. Consistent with this, during unwinding of both χ°- and χ + -DNA, adozelesin induced the formation of apparent χ-specific fragments. These are indicated as apparent, as we do not attribute their formation to an enzyme reaching χ and eliciting the anticipated response, i.e. the production of the anticipated and correct-sized χ-specific fragments. Instead, they result from adozelesin-induced modification of RecBCD. The DNA substrates used in χ0 and χ+ experiments were identical in sequence, and were constructed using EcoRI and BamHI, restriction enzymes, respectively (see Material and Methods). Since distinct sized apparent χ-specific fragments were produced using each of these substrates, this implies that RecBCD pausing leading to modification occurred at the sites in each substrate, likely determined by the sites of modification by adozelesin. The fragment sizes are different, as the substrates were cleaved with different restriction enzymes. The two models may not be mutually exclusive, since it is conceivable that both could be occurring. Additional work will be required to map precisely the sites of trapping and/or pseudo-χ recognition, to ascertain whether they are the same. The ability of adozelesin to trap a translocating enzyme may have

DNA Alkylators Inhibit RecBCD Enzyme

important ramifications in vivo. Here, only one end of dsDNA may be exposed, so that once an enzyme such as RecBCD binds and initiates translocation and DNA unwinding, it may become entrapped permanently. This will arise as no free end is available to permit entry of a second RecBCD molecule to facilitate displacement of the trapped enzyme. It is conceivable, however, that the distortion induced by adozelesin in dsDNA may decrease the processivity of the translocating RecBCD enzyme so that it dissociates more frequently from adducted DNA relative to unmodified DNA. Premature termination from the DNA would also produce partially unwound intermediates. Although this may be occurring it does not explain the biphasic time-courses. The loss of RecBCD activity during subsequent rounds of DNA unwinding under catalytic conditions is consistent with transient, adozelesin-induced enzyme trapping on the DNA (Table 2). The delayed appearance of ssDNA under stoichiometric conditions at later times in reactions is also consistent with enzyme entrapment. Therefore, we attribute the formation of stable intermediates visualized in agarose gels to trapping of the translocating RecBCD enzyme (although we cannot exclude pseudo-χ recognition entirely), as observed for other DNA helicases.8 Hedamycin was the least effective agent used in this study. Although this agent intercalates into DNA in the major groove,58 it does interact with the minor groove but to a lesser extent.60 Both hedamycin and Et743 alter the structure of the duplex significantly, protrude partially from DNA and increase the melting temperature of the duplex. Et743 interacts exclusively with the minor groove and is the more potent inhibitor, suggesting that modification of the minor groove is more effective in disrupting RecBCD enzyme-catalyzed DNA unwinding than is intercalation between base-pairs. Although binding of hedamycin produces a 20 deg. C increase in the DNA melting temperature,56 modification of dsDNA by hedamycin did not inhibit the progress of RecBCD significantly, as only a modest reduction in unwinding rate and little to no effect on extent was observed. Significant inhibition did occur, however, at elevated drug concentrations where, on average, one drug molecule would be encountered every 5 bp (Figure 7). Similar results for intercalating dye molecules (e.g. ethidium bromide) on RecBCD have been observed using fluorescence-based assays.73 As for hedamycin, little or no inhibition is observed at low concentrations of ethidium bromide, while significant inhibition requires elevated concentrations of dye. The effects of hedamycin on the unwinding of DNA by RecBCD are complicated by the presence of free, unreacted drug molecules. It was the only agent used here where unbound drug molecules affected enzyme activity, as a greater level of inhibition of the rate of unwinding was observed using non-precipitated DNA, which contained detectable levels of free drug molecules (Table 1). Even though free adozelesin was present following treatment with the drug, as shown in the heat-induced, DNA strand cleavage

DNA Alkylators Inhibit RecBCD Enzyme

assay, free adozelesin did not affect either SSB or RecBCD (data not shown; and see Table 1). Incubation of RecBCD with hedamycin in the absence of DNA resulted in slower rates of unwinding (e.g. at 30 μM a 50% decrease compared to the control was observed, data not shown). In addition, free hedamycin inactivated SSB as extended incubations of drug and protein resulted in decreased ability of SSB to bind to unmodified ssDNA (data not shown). Consequently, experiments using hedamycin were done using drug-treated and ethanol-precipitated dsDNA. The rate of unwinding of ethanol-precipitated dsDNA was reduced by 35% relative to the control, indicating that DNA-bound hedamycin is able to slow the progress of the translocating RecBCD enzyme, albeit not very effectively. The intercalation of hedamycin into dsDNA resulted in a novel and unanticipated effect on the nuclease activity and χ-recognition of RecBCD. Unlike adozelesin or Et743, hedamycin did not completely prevent the recognition and response of RecBCD to χ. Instead, formation of the downstream, top-strand χ-specific fragment still occurred, albeit at a level reduced by twofold compared to the control reaction (Figure 8, top-strand, (ii)). As both the levels of full-length ssDNA and that of the bottom-strand, upstream χ-specific fragment were reduced significantly, this suggests that the bottom-strand nuclease activity was activated constitutively. A gating mechanism has been proposed to control the access of each unwound single strand of DNA to the single nuclease site in RecB.15 Thus, the alkylation of the DNA by hedamycin perturbs the gating mechanism so that the 5′-terminated strand relative to the entry point of the enzyme (the bottom strand on which RecD translocates) is exposed more frequently to the nuclease domain. Consequently, before interacting with χ, both strands of DNA are cleaved frequently. As the ability of the enzyme to recognize and respond to χ is largely unaffected, nuclease activity of the top strand is still attenuated, resulting in the production of the downstream, topstrand χ-specific fragment. Formation of the bottomstrand, upstream χ-specific fragment does not occur, since it was prematurely degraded. The ability of minor groove binding agents to inhibit the activity of DNA helicases is not without precedence. Similar effective inhibition of the unwinding of DNA by the BLM and Werner's DNA helicases by the minor groove binder distamycin A has been observed.13 Further, and similar to RecBCD, intercalating agents such as ethidium bromide and actinomycin D had little effect on any of these proteins.13 These results argue that for several DNA helicases, interaction of the protein with the minor groove ahead of the translocating enzyme as observed for RecBCD, plays a critical role in the mechanism of DNA unwinding. This, however, is not a general rule applicable to all DNA helicases, as the E. coli UvrD enzyme is inhibited by intercalating agents and not by minor groove binding agents.9,13 During translocation and DNA unwinding, RecBCD hydrolyzes two to three ATP molecules per base-pair unwound.19,70 Each agent decreased both

915 the rate of DNA unwinding, and concomitantly, the rate of ATP hydrolysis (Table 3). For each inhibitor, the reduction in DNA unwinding paralleled that of the rate of ATP hydrolysis. For Et743, the most potent inhibitor, only a twofold decrease in the ATP utilization efficiency was observed. Thus, these inhibitors slow the progress of the translocating enzyme without perturbing significantly the allosteric interaction that activates the enzyme for ATP hydrolysis. The fluorescence-based assay requires SSB binding to unwound ssDNA to detect DNA unwinding. This results in the intrinsic fluorescence of SSB being quenched as RecBCD translocates and unwinds the duplex. As the agarose gel assay demonstrates that drug-modified DNA is being unwound, and that a terminally stalled enzyme molecule is not simply removing the 5′-label, thereby giving the appearance of unwinding, the elevated level of inhibition observed in the fluorescence assays must be due to inhibition of SSB binding to the modified and unwound ssDNA. This results from the increased nuclease activity of RecBCD producing small nucleolytic fragments that are not bound efficiently by SSB. Alternatively, the addition of the alkyl groups may preclude binding of SSB to nucleolytic fragments to which it may otherwise bind. The outcome, however, is the same; i.e. SSB binding resulting in its intrinsic fluorescence being quenched does not occur efficiently. During the course of performing the fluorescence assays, mixtures of drug-modified DNA, buffer and SSB were kept on ice before being placed into the spectrofluorimeter. Reaction mixes with hedamycintreated dsDNA kept on ice for extended lengths of time had lower initial fluorescence, and did not accurately report dsDNA unwinding by RecBCD (data not shown). When reactions were repeated, but with SSB added immediately before transferring mixtures to cuvettes, unwinding was reported accurately. This suggests that free hedamycin molecules can inactivate or otherwise inhibit SSB protein. DNA-bound hedamycin was able to inhibit the translocating RecBCD enzyme, as evidenced by assays using ethanol-precipitated and drug-modified DNA. Therefore, an additional means of exerting an effect on actively replicating cells, may be to inhibit binding of single-stranded DNA-binding proteins, thereby exposing the DNA to attack by nucleases. In summary, we have demonstrated that RecBCD is an excellent model enzyme to evaluate the effects of DNA-binding agents. The effects of DNA-binding/ modifying agents on DNA helicase, nuclease, and ATPase, and the ability to read and respond to specific sequences can be assessed in a single enzyme. Further, these effects can be correlated with the crystal structure of the enzyme, further contributing to the insight obtained in studies with this enzyme. The results show that the mechanism of inhibition of the translocating RecBCD enzyme by these three alkylating agents is complex and can be attributed to a combination of factors. First, they interact with DNA in the minor groove and distort the structure of the duplex. This impedes the progress of dsDNA into the holoenzyme.

916 Second, modification of the DNA results in increased stability of the duplex, so that the process of strand separation is affected. Third, the covalent attachment of a bulky adduct affects the interaction of the enzyme with duplex DNA and the passage of unwound single strands of DNA through channels within RecC. These factors combine to slow the progress of RecBCD through the DNA, providing the nuclease domain an increased opportunity to interact with ssDNA, which it consequently cleaves more frequently. As the progress of the enzyme is markedly slowed, and the allosteric interaction that activates the enzyme for ATP hydrolysis is largely unaffected, the rate of ATP hydrolysis parallels the unwinding rate. That is, both are reduced by similar amounts. Consistent with previous reports, the agents tested can be separated into intercalating agents and minor groove binding agents. For RecBCD, disruption of the interactions with the minor groove demonstrates an important role of these interactions in the biochemical mechanism of translocation and DNA unwinding by this enzyme.

Materials and Methods Reagents Et743 and hedamycin were provided by NCI, while adozelesin was a generous gift from Pharmacia, Kalamazoo, MI. Stock solutions of 4 mM adozelesin and 10 mM Et743 were made by dissolving the drugs in dimethyl sulfoxide (DMSO), while hedamycin was dissolved at 10 mM in water. All drugs were stored at −20 °C and protected from light. [γ-32P]ATP (10 μCi/μl; 6000 Ci/mmol) and Sephadex G-50 columns were purchased from Amersham Bioscience (Piscataway, NJ). SYBR-green was from Molecular Probes (Eugene, OR). Restriction endonucleases, bacteriophage T4 polynucleotide kinase (PNK) and the GeneRuler™ DNA Ladder Mix, were purchased from Fermentas Inc. (Hanover, MD). Calf intestine alkaline phosphatase (CIP) was purchased from Roche Diagnostic Corp (Indianapolis, IN). All other chemicals were of reagent grade. Lactate dehydrogenase and pyruvate kinase were purchased from Sigma as suspensions in ammonium sulfate. ATP was purchased from Amersham Pharmacia and used to prepare a concentrated stock of 1 M Tris–HCl (pH 7.5). The concentration of ATP was determined by using ε=15.4×103 M−1 cm−1.

DNA Alkylators Inhibit RecBCD Enzyme using ε = 30,000 M−1 cm−1. The site size of SSB was determined to be ten nucleotides per monomer by monitoring the quenching of the intrinsic fluorescence of SSB that occurs on binding to ssDNA, as described.76 Plasmid DNA Supercoiled plasmid DNA (pPB12 or pSVA03) was isolated using a Qiagen Mega Kit. Purified DNA was stored in TE buffer (10 mM Tris–HCl (pH 7.5), 1 mM EDTA) at −20 °C. The nucleotide concentration of dsDNA was determined by measuring the absorbance at 260 nm, using ε = 6,500 M−1 cm−1. For all RecBCD reactions, the same substrate DNA, pPB12, was used in attempts to minimize non-χ, sequence-specific effects. This DNA contains a tandem array of three repeated χ sequences with 10 bp of DNA between each. Cleavage by EcoRI orients the χ-array 50 bp from one end of the DNA, in the correct orientation for χ-recognition by the translocating enzyme to occur. However, when positioned this close to an end, χ is not recognized efficiently by the translocating RecBCD enzyme (P.B. unpublished results; and data not shown). Thus, EcoRI-cut pPB12 DNA was considered throughout this study as χ0-DNA. To position the χ-array so that efficient χ-recognition by the translocating RecBCD enzyme could occur, supercoiled DNA was restricted with BamHI. This DNA is designated as χ+-DNA, with the χ-sequence positioned approximately 1100 bp from one end (Figure 1(b)). Linearized pPB12 was dephosphorylated with calf intestine alkaline phosphatase, heat-inactivated at 75 °C for 10 min in the presence of 5 mM EDTA (pH 8.0) extracted with an equal volume of phenol and precipitated in ethanol. The resuspended DNA was 5′ end-labeled with [32P] γ-ATP using T4 polynucleotide kinase. The radioactively labeled DNA was separated from unincorporated nucleotides using Sephadex G-50 columns. Drug treatment of dsDNA

Proteins

EcoRI or BamHI-linearized pPB12 was incubated with increasing concentrations of drug in separate reactions or with drug solvent at 37 °C for 1 h, in a total volume of 20 μl. The DNA was either used directly for helicase assays or stored overnight at −20 °C. Alternatively, dsDNA was precipitated from free drug with three volumes of ice-cold ethanol, 2 μg/ml of glycogen, 0.3 mM sodium acetate overnight at −20 °C. Following centrifugation and drying, the DNA was resuspended in 20 μl of double distilled water.

Purification of biotinylated RecBCD enzyme

Heat-induced, DNA strand cleavage assay

The growth of cells, the induction of expression of genes and protein purification were as described.74 The concentration of purified enzyme was determined by measuring the absorbance at 280 nm using ε = 40,000 M−1cm−1. The amount of active RecBCD enzyme (80%) was determined using the fluorescence-based helicase assay as described.19 The active concentration of RecBCD is given for all reactions. SSB E. coli SSB was purified from strain K12ΔH1Δtrp as described.75 The concentration of purified SSB was determined by measuring the absorbance at 280 nm

This assay was used to assess the amount of either adozelesin or hedamycin bound to dsDNA. It is modified from Reynolds et al.68 The assay is performed as follows: the first plasmid, pPB12 (4100 bp in length) was incubated with 0.3 μM drug (nine drugs molecules per DNA molecule) for 60 min and precipitated in ethanol. The modified DNA was resuspended in distilled water, the second plasmid was added (pSVA03; 5200 bp in length) and the incubation continued. Thereafter, the plasmid mixture was heated to 70 °C to induce DNA breaks at the site of modification by the drug molecules. The mixture was mixed with gel-loading dye and subjected to electrophoresis in agarose gels. The relative amounts of

917

DNA Alkylators Inhibit RecBCD Enzyme supercoiled DNA remaining were determined following SYBR-green staining of the gel. Spectrofluorimetric helicase assay The helicase activity of RecBCD was assayed by observing quenching of the intrinsic fluorescence of SSB upon binding to ssDNA.19 Assays (500 μl) were done at 20 °C and contained 25 mM Tris–acetate (pH 7.5), 2 mM magnesium acetate, 1 mM DTT, 1 mM ATP, 1 μM SSB, 2.4 nM DNA (molecules) and 2 nM RecBCD. A portion (475 μl) of a mix containing reaction buffer, ATP and SSB was added to the drug-treated or unmodified DNA and incubated for 2 min before reaction initiation. Reactions were initiated by the addition of enzyme and monitored for 5 min. The fluorescence data were collected as a function of time using a Varian Cary Eclipse fluorescence spectrophotometer equipped with a multi-cell holder that was connected to a PCB150 Peltier-controlled water circulator that maintained temperature within ±0.5 deg. C. The excitation and emission wavelengths were set at 290 nm and 340 nm, respectively; the excitation slit-width was 2.5 nm and the emission slit-width was 20 nm. All rates are reported in nM bp s−1 nM−1 RecBCD.19 Agarose gel helicase assay Reactions were done at room temperature (22–24 °C) in a volume of 60 μl and contained 25 mM Tris–acetate (pH 7.5), 1 mM DTT, 2 mM magnesium acetate, 1 mM ATP, 10 μM SSB, DNA at 95 μM (in nucleotides) and 18 nM RecBCD. At the indicated time-points, DNA aliquots were transferred to fresh tubes containing 2 μl of stop mix (5 mM EDTA (pH 8.0), 1% (w/v) SDS, and 50 μg/ml of proteinase K, 20% (v/v) Ficoll 400, 0.25% (w/v) bromophenol blue and 0.25% (w/v) xylene cyanol). After collecting all timepoints, DNA was loaded onto 1% (w/v) agarose gels and subjected to electrophoresis in TAE buffer for 17 h at 35 V. Following electrophoresis, gels were vacuum-dried and exposed to Phosphorimager screens. Dried gels were quantified using Image Quant version 5.2 (Molecular Dynamics). The initial rates of DNA unwinding were calculated from the slopes of tangent lines fitted to the early portion of the time-course. In reactions where biphasic behaviour was observed, the rate of unwinding was calculated from tangent lines fitted to these regions of the time-courses. The extent of DNA unwinding was determined by comparing the amount of dsDNA substrate in the gel at time zero (Figure 3(b)) to the dsDNA signal remaining at 1200 s after initiating the reaction with RecBCD. Spectrophotometric ATPase assay The assay used to monitor ATP hydrolysis is a spectrophotometric assay that couples the hydrolysis of ATP to a decrease in the absorbance of NADH.77 Reactions (200 μl) contained 25 mM Tris–acetate (pH 7.5), 2 mM magnesium acetate, 1 mM ATP, 1 mM DTT, 3 mM phosphoenolpyruvate, 100 μg/ml of NADH and approximately 40 units/ ml of each: lactate dehydrogenase and pyruvate kinase, 40 μM nt of dsDNA (pPB12, restricted with EcoRI), 8 nM RecBCD and 4 μM SSB protein (enough to saturate all ssDNA). The absorbance data were collected as a function of time using a Varian Cary 50 spectrophotometer equipped with an 18-cell holder that was connected to a PCB150 Peltier-controlled water circulator that main-

tained the temperature within ±0.5 deg. C. Initial rates (in μM min−1) were determined by fitting a straight line tangent to the data, and multiplying the slope of this line by 160.78 The initial rate of ATP hydrolysis was determined from the slope at the linear portion of the timecourse. The assays were performed at 20 °C and initiated by the addition of RecBCD enzyme to a cuvette containing all the other components.

References 1. Anthoney, D. A. & Twelves, C. J. (2001). DNA: still a target worth aiming at? A review of new DNAinteractive agents. Am. J. Pharmacogenomics, 1, 67–81. 2. Hurley, L. H. (2002). DNA and its associated processes as targets for cancer therapy. Nature Rev. Cancer, 2, 188–200. 3. Izbicka, E. & Tolcher, A. W. (2004). Development of novel alkylating drugs as anticancer agents. Curr. Opin. Investig. Drugs, 5, 587–591. 4. Lohman, T. M. & Bjornson, K. P. (1996). Mechanisms of helicase-catalyzed DNA unwinding. Annu. Rev. Biochem. 65, 169–214. 5. Hall, M. C. & Matson, S. W. (1999). Helicase motifs: the engine that powers DNA unwinding. Mol. Microbiol. 34, 867–877. 6. Patel, S. S. & Picha, K. M. (2000). Structure and function of hexameric helicases. Annu. Rev. Biochem. 69, 651–697. 7. Delagoutte, E. & von Hippel, P. H. (2003). Helicase mechanisms and the coupling of helicases within macromolecular machines. Part II: Integration of helicases into cellular processes. Quart. Rev. Biophys. 36, 1–69. 8. Villani, G. & Tanguy, L. G. (2000). Interactions of DNA helicases with damaged DNA: possible biological consequences. J. Biol. Chem. 275, 33185–33188. 9. George, J. W., Ghate, S., Matson, S. W. & Besterman, J. M. (1992). Inhibition of DNA helicase II unwinding and ATPase activities by DNA-interacting ligands. Kinetics and specificity. J. Biol. Chem. 267, 10683–10689. 10. Tuteja, N., Phan, T. N., Tuteja, R., Ochem, A. & Falaschi, A. (1997). Inhibition of DNA unwinding and ATPase activities of human DNA helicase II by chemotherapeutic agents. Biochem. Biophys. Res. Commun. 236, 636–640. 11. Bachur, N. R., Yu, F., Johnson, R., Hickey, R., Wu, Y. & Malkas, L. (1992). Helicase inhibition by anthracycline anticancer agents. Mol. Pharmacol. 41, 993–998. 12. Bachur, N. R., Lun, L., Sun, P. M., Trubey, C. M., Elliott, E. E., Egorin, M. J. et al. (1998). Anthracycline antibiotic blockade of SV40 T antigen helicase action. Biochem. Pharmacol. 55, 1025–1034. 13. Brosh, R. M., Jr, Karow, J. K., White, E. J., Shaw, N. D., Hickson, I. D. & Bohr, V. A. (2000). Potent inhibition of Werner and Bloom helicases by DNA minor groove binding drugs. Nucl. Acids Res. 28, 2420–2430. 14. Kowalczykowski, S. C., Dixon, D. A., Eggleston, A. K., Lauder, S. D. & Rehrauer, W. M. (1994). Biochemistry of homologous recombination in Escherichia coli. Microbiol. Rev. 58, 401–465. 15. Singleton, M. R., Dillingham, M. S., Gaudier, M., Kowalczykowski, S. C. & Wigley, D. B. (2004). Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature, 432, 187–193. 16. Kuzminov, A. (1999). Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. Microbiol. Mol. Biol. Rev. 63, 751–813. 17. Dillingham, M. S., Spies, M. & Kowalczykowski, S. C.

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18. 19.

20.

21.

22.

23.

24. 25.

26.

27.

28.

29.

30.

31. 32.

33.

34.

(2003). RecBCD enzyme is a bipolar DNA helicase. Nature, 423, 893–897. Taylor, A. F. & Smith, G. R. (2003). RecBCD enzyme is a DNA helicase with fast and slow motors of opposite polarity. Nature, 423, 889–893. Roman, L. J. & Kowalczykowski, S. C. (1989). Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay. Biochemistry, 28, 2863–2873. Roman, L. J., Eggleston, A. K. & Kowalczykowski, S. C. (1992). Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme. J. Biol. Chem. 267, 4207–4214. Bianco, P. R., Brewer, L. R., Corzett, M., Balhorn, R., Yeh, Y., Kowalczykowski, S. C. & Baskin, R. J. (2001). Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature, 409, 374–378. Dixon, D. A. & Kowalczykowski, S. C. (1993). The recombination hotspot chi is a regulatory sequence that acts by attenuating the nuclease activity of the E. coli RecBCD enzyme. Cell, 73, 87–96. Eichler, D. C. & Lehman, I. R. (1977). On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli. J. Biol. Chem. 252, 499–503. Smith, G. R., Kunes, S. M., Schultz, D. W., Taylor, A. & Triman, K. L. (1981). Structure of chi hotspots of generalized recombination. Cell, 24, 429–436. Bianco, P. R. & Kowalczykowski, S. C. (1997). The recombination hotspot chi is recognized by the translocating RecBCD enzyme as the single strand of DNA containing the sequence 5′-GCTGGTGG-3′. Proc. Natl Acad. Sci. USA, 94, 6706–6711. Spies, M., Bianco, P. R., Dillingham, M. S., Handa, N., Baskin, R. J. & Kowalczykowski, S. C. (2003). A molecular throttle: the recombination hotspot chi controls DNA translocation by the RecBCD helicase. Cell, 114, 647–654. Anderson, D. G. & Kowalczykowski, S. C. (1997). The recombination hot spot chi is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme. Genes Dev. 11, 571–581. Anderson, D. G. & Kowalczykowski, S. C. (1997). The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a chiregulated manner. Cell, 90, 77–86. Arnold, D. A., Bianco, P. R. & Kowalczykowski, S. C. (1998). The reduced levels of chi recognition exhibited by the RecBC1004D enzyme reflect its recombination defect in vivo. J. Biol. Chem. 273, 16476–16486. Arnold, D. A., Handa, N., Kobayashi, I. & Kowalczykowski, S. C. (2000). A novel, 11 nucleotide variant of chi, chi*: one of a class of sequences defining the Escherichia coli recombination hotspot chi. J. Mol. Biol. 300, 469–479. Bianco, P. R. & Kowalczykowski, S. C. (2000). Translocation step size and mechanism of the RecBC DNA helicase. Nature, 405, 368–372. Cobuzzi, R. J., Burhans, W. C. & Beerman, T. A. (1996). Inhibition of initiation of simian virus 40 DNA replication in infected BSC-1 cells by the DNA alkylating drug adozelesin. J. Biol. Chem. 271, 19852–19859. Tu, L. C., Melendy, T. & Beerman, T. A. (2004). DNA damage responses triggered by a highly cytotoxic monofunctional DNA alkylator, hedamycin, a pluramycin antitumor antibiotic. Mol. Cancer Ther. 3, 577–585. Dziegielewska, B., Kowalski, D. & Beerman, T. A. (2004). SV40 DNA replication inhibition by the

DNA Alkylators Inhibit RecBCD Enzyme

35.

36.

37.

38. 39.

40.

41. 42.

43.

44.

45. 46.

47.

48.

49.

monofunctional DNA alkylator Et743. Biochemistry, 43, 14228–14237. Jin, S. G., Choi, J. H., Ahn, B., O'Connor, T. R., Mar, W. & Lee, C. S. (2001). Excision repair of adozelesin-N3 adenine adduct by 3-methyladenine-DNA glycosylases and UvrABC nuclease. Mol. Cells, 11, 41–47. Zewail-Foote, M., Li, V. S., Kohn, H., Bearss, D., Guzman, M. & Hurley, L. H. (2001). The inefficiency of incisions of ecteinascidin 743-DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repairdependent toxicities of this antitumor agent. Chem. Biol. 8, 1033–1049. Boyd, F. L., Stewart, D., Remers, W. A., Barkley, M. D. & Hurley, L. H. (1990). Characterization of a unique tomaymycin-d(CICGAATTCICG)2 adduct containing two drug molecules per duplex by NMR, fluorescence, and molecular modeling studies. Biochemistry, 29, 2387–2403. Weiland, K. L. & Dooley, T. P. (1991). In vitro and in vivo DNA bonding by the CC-1065 analogue U-73975. Biochemistry, 30, 7559–7565. Hurley, L. H., Warpehoski, M. A., Lee, C. S., McGovren, J. P., Scahill, T. A., Kelly, R. C. et al. (1990). Sequence specificity of DNA alkylation by the unnatural enantiomer of CC-1065 and its synthetic analogues. J. Am. Chem. Soc. 112, 4633–4649. Lee, C. S., Sun, D., Kizu, R. & Hurley, L. H. (1991). Determination of the structural features of (+)-CC1065 that are responsible for bending and winding of DNA. Chem. Res. Toxicol. 4, 203–213. Cameron, L. & Thompson, A. S. (2000). Determination of the structural role of the linking moieties in the DNA binding of adozelesin. Biochemistry, 39, 5004–5012. Sun, D. & Hurley, L. H. (1992). Inhibition of T4 DNA ligase activity by (+)-CC-1065: demonstration of the importance of the stiffening and winding effects of (+)CC-1065 on DNA. Anticancer Drug Des. 7, 15–36. Sun, D. & Hurley, L. H. (1992). Structure-activity relationships of (+)-CC-1065 analogues in the inhibition of helicase-catalyzed unwinding of duplex DNA. J. Med. Chem. 35, 1773–1782. Sun, D. & Hurley, L. H. (1992). Effect of the (+)-CC1065-(N3-adenine)DNA adduct on in vitro DNA synthesis mediated by Escherichia coli DNA polymerase. Biochemistry, 31, 2822–2829. Zewail-Foote, M. & Hurley, L. H. (1999). Ecteinascidin 743: a minor groove alkylator that bends DNA toward the major groove. J. Med. Chem. 42, 2493–2497. Sakai, R., Jares-Erijman, E. A., Manzanares, I., Silva, M., Elipe, V. & Rinehart, K. L. (1996). Ecteinascidins: Putative biosynthetic precursors and absolute stereochemistry. J. Am. Chem. Soc. 118, 9017–9023. Pommier, Y., Kohlhagen, G., Bailly, C., Waring, M., Mazumder, A. & Kohn, K. W. (1996). DNA sequence- and structure-selective alkylation of guanine N2 in the DNA minor groove by ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia turbinata. Biochemistry, 35, 13303–13309. Zewail-Foote, M. & Hurley, L. H. (2001). Differential rates of reversibility of ecteinascidin 743-DNA covalent adducts from different sequences lead to migration to favored bonding sites. J. Am. Chem. Soc. 123, 6485–6495. Hurley, L. H. & Zewail-Foote, M. (2001). The antitumor agent ecteinascidin 743: characterization of its covalent DNA adducts and chemical stability. Advan. Expt. Med. Biol. 500, 289–299.

DNA Alkylators Inhibit RecBCD Enzyme 50. D'Incalci, M., Erba, E., Damia, G., Galliera, E., Carrassa, L., Marchini, S. et al. (2002). Unique features of the mode of action of ET-743. Oncologist, 7, 210–216. 51. van Kesteren, C., de Vooght, M. M., Lopez-Lazaro, L., Mathot, R. A., Schellens, J. H., Jimeno, J. M. & Beijnen, J. H. (2003). Yondelis (trabectedin, ET-743): the development of an anticancer agent of marine origin. Anticancer Drugs, 14, 487–502. 52. Bonfanti, M., La Valle, E., Fernandez Sousa Faro, J. M., Faircloth, G., Caretti, G., Mantovani, R. & D'Incalci, M. (1999). Effect of ecteinascidin-743 on the interaction between DNA binding proteins and DNA. Anticancer Drug Des. 14, 179–186. 53. Minuzzo, M., Marchini, S., Broggini, M., Faircloth, G., D'Incalci, M. & Mantovani, R. (2000). Interference of transcriptional activation by the antineoplastic drug ecteinascidin-743. Proc. Natl Acad. Sci. USA, 97, 6780–6784. 54. Takebayashi, Y., Pourquier, P., Zimonjic, D. B., Nakayama, K., Emmert, S., Ueda, T. et al. (2001). Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotideexcision repair. Nature Med. 7, 961–966. 55. Bradner, W. T., Heinemann, B. & Gourevitch, A. (1966). Hedamycin, a new antitumor antibiotic. II. Biological properties. Antimicrobial. Agents Chemother. 6, 613–618. 56. White, H. L. & White, J. R. (1969). Hedamycin and rubiflavin complexes with deoxyribonucleic acid and other polynucleotides. Biochemistry, 8, 1020–1042. 57. Pavlopoulos, S., Bicknell, W., Wickham, G. & Craik, D. J. (1999). Characterization of the sequential noncovalent and covalent interactions of the antitumour antibiotic hedamycin with double stranded DNA by NMR spectroscopy. J. Mol. Recogn. 12, 346–354. 58. Hansen, M., Yun, S. & Hurley, L. (1995). Hedamycin intercalates the DNA helix and, through carbohydrate-mediated recognition in the minor groove, directs N7-alkylation of guanine in the major groove in a sequence-specific manner. Chem. Biol. 2, 229–240. 59. Pavlopoulos, S., Bicknell, W., Craik, D. J. & Wickham, G. (1996). Structural characterization of the 1:1 adduct formed between the antitumor antibiotic hedamycin and the oligonucleotide duplex d(CACGTG)2 by 2D NMR spectroscopy. Biochemistry, 35, 9314–9324. 60. Sun, D., Hansen, M., Clement, J. J. & Hurley, L. H. (1993). Structure of the altromycin B (N7-guanine)DNA adduct. A proposed prototypic DNA adduct structure for the pluramycin antitumor antibiotics. Biochemistry, 32, 8068–8074. 61. Joel, P. B. & Goldberg, I. H. (1970). The inhibition of RNA and DNA polymerases by hedamycin. Biochim. Biophys. Acta, 224, 361–370. 62. Chiang, S. Y., Welch, J., Rauscher, F. J., III & Beerman, T. A. (1994). Effects of minor groove binding drugs on the interaction of TATA box binding protein and TFIIA with DNA. Biochemistry, 33, 7033–7040. 63. Welch, J. J., Rauscher, F. J., III & Beerman, T. A. (1994). Targeting DNA-binding drugs to sequence-specific transcription factor. DNA complexes. Differential effects of intercalating and minor groove binding drugs. J. Biol. Chem. 269, 31051–31058.

919 64. Villani, G., Cazaux, C., Pillaire, M. J. & Boehmer, P. (1993). Effects of a single intrastrand d(GpG) platinum adduct on the strand separating activity of the Escherichia coli proteins RecB and RecA. FEBS Letters, 333, 89–95. 65. Karu, A. E. & Linn, S. (1972). Uncoupling of the recBC ATPase from DNase by DNA crosslinked with psoralen. Proc. Natl Acad. Sci. USA, 69, 2855–2859. 66. Eggleston, A. K. & Kowalczykowski, S. C. (1993). The mutant recBCD enzyme, recB2109CD enzyme, has helicase activity but does not promote efficient joint molecule formation in vitro. J. Mol. Biol. 231, 621–633. 67. Eggleston, A. K. & Kowalczykowski, S. C. (1993). Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks chispecific, but not non-specific, nuclease activity. J. Mol. Biol. 231, 605–620. 68. Reynolds, V. L., Molineux, I. J., Kaplan, D. J., Swenson, D. H. & Hurley, L. H. (1985). Reaction of the antitumor antibiotic CC-1065 with DNA. Location of the site of thermally induced strand breakage and analysis of DNA sequence specificity. Biochemistry, 24, 6228–6237. 69. Jin, S. G., Choi, J. H., Ahn, B., O'Connor, T. R., Mar, W. & Lee, C. S. (2001). Excision repair of adozelesin-N3 adenine adduct by 3-methyladenine-DNA glycosylases and UvrABC nuclease. Mol. Cells, 11, 41–47. 70. Roman, L. J. & Kowalczykowski, S. C. (1989). Characterization of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydrolysis to the unwinding of duplex DNA. Biochemistry, 28, 2873–2881. 71. Bianco, P. R. & Kowalczykowski, S. C. (2000). Translocation step size and mechanism of the RecBC DNA helicase. Nature, 405, 368–372. 72. Cheng, K. C. & Smith, G. R. (1987). Cutting of chi-like sequences by the RecBCD enzyme of Escherichia coli. J. Mol. Biol. 194, 747–750. 73. Eggleston, A. K., Rahim, N. A. & Kowalczykowski, S. C. (1996). A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands. Nucl. Acids Res. 24, 1179–1186. 74. Handa, N., Bianco, P. R., Baskin, R. J. & Kowalczykowski, S. (2005). Direct visualization of RecBCD v of the RecD Motor after X recognition. Mol. Cells, 17, 745–750. 75. Lohman, T. M., Green, J. M. & Beyer, R. S. (1986). Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under lambda PL control. Biochemistry, 25, 21–25. 76. Lohman, T. M. & Overman, L. B. (1985). Two binding modes in Escherichia coli single strand binding proteinsingle stranded DNA complexes. Modulation by NaCl concentration. J. Biol. Chem. 260, 3594–3603. 77. Kowalczykowski, S. C., Clow, J. & Krupp, R. A. (1987). Properties of the duplex DNA-dependent ATPase activity of Escherichia coli RecA protein and its role in branch migration. Proc. Natl Acad. Sci. USA, 84, 3127–3131. 78. Kreuzer, K. N. & Jongeneel, C. V. (1983). Escherichia coli phage T4 topoisomerase. Methods Enzymol. 100, 144–160.

Edited by K. Morikawa (Received 23 March 2006; received in revised form 26 June 2006; accepted 28 June 2006) Available online 2 August 2006