The capture of a DNA double helix by an ATP-dependent protein clamp: A key step in DNA transport by type II DNA topoisomerases

Cell, Vol. 71, 833-840,

November

27, 1992, Copyright

0 1992 by Cell Press

The Capture of a DNA Double Helix by an ATP-Dependent Protein Clamp: A Key Step in DNA Transport by Type II DNA Topoisomerases Joaquim Rota and James Department of Biochemistry Harvard University Cambridge, Massachusetts

C. Wang and Molecular

Biology

02138

Summary The binding of linear and circular forms of DNA to yeast DNA topoisomerase II or its complex with AMPPNP, the nonhydrolyzable S,y-imido analog of ATP, wascarried out to probe the ATP analog-induced conformational change of the enzyme. Binding of the ATP analog is shown to convert the enzyme to a circular clamp with an annulet, through which only a linear DNA can pass; subsequent circularization of the bound linear DNA forms a salt-stable catenane between the protein circular clamp and the DNA ring. Analysis of catenane formation between a small DNA ring originally bound to the topoisomerase and a large DNA ring subsequently added, under conditions such that the two do not exchange, supports a model in which a second DNA double-helix can enter the open jaws of a DNAbound protein clamp, and the closure of the jaws upon ATP-binding traps the second duplex and transports it through an enzyme-operated gate in the first DNA duplex. Introduction Type II DNA topoisomerases are found in all living organisms, and members of this family include bacterial gyrase (bacterial DNAtopoisomerase II), bacterial DNA topoisomerase IV, eukaryotic DNA topoisomerase II, and T-even phage encoded DNA topoisomerases (for a recent review, see Wang, 1991). All type II DNA topoisomerases are evolutionarily related (Lynn et al., 1986; Wyckoff et al., 1989; Huang, 1990; Uemuraetal., 1986), andtheycatalyze ATPdependent catenationldecatenation, knotting/unknotting, or alteration of the linking numbers of double-stranded DNA rings. A type II DNA topoisomerase performs these topological transformations by transporting one doublestranded DNA segment through an enzyme-mediated transient double-stranded break in another. Following this DNA passage event, the gate in the duplex DNA is closed through rejoining of the severed DNA strands by the enzyme (Brown and Cozzarelli, 1979; Liu et al., 1980). Biochemical and genetic experiments indicate that the type II DNA topoisomerases are essential in the segregation of newly replicated pairs of intertwined chromosomes (Holm et al., 1985; Uemura and Yanagida, 1986); they are also normally involved in a number of other vital transactions of DNA, including chromosomal condensationldecondensation and the modulation of the state of supercoiling of intracellular DNA (reviewed in Yanagida and Wang, 1987; Yanagida and Sternglanz, 1990). Interest in this class of enzymes has been further stimulated through their identifi-

cation as the targets of a large number of antimicrobial and anticancer drugs (reviewed in Drlica and France, 1988; Liu, 1989) as well as natural toxins (Vizan et al., 1991; Miki et al., 1992). Mechanistically, one of the most fascinating aspects of type II DNA topoisomerases is their coupling of ATP usage to the manipulation of DNA. Unlike other complex ATPdependent multiprotein systems, a type II DNA topoisomerase acts as a single molecule with a well-characterized quaternary structure in its coupling of ATP binding and hydrolysis to the breakage, passage, and rejoining of DNA. The enzyme from the budding yeast Saccharomyces cerevisiae, for example, acts as a homodimer of a 170 kd polypeptide (Goto and Wang, 1982). A comparison of the amino acid sequences shows that the N-terminal 400 amino acids of the yeast enzyme is highly homologous to the ATPase domain of Escherichia coli gyrase B subunit, the three-dimensional structure of which has been determined recently (Wigley et al., 1991). The finding that changing Gly144 of the yeast enzyme to isoleucine abolishes its ATPase activity has provided further evidence that the ATPase site of the eukaryotic enzyme resides in the amino-terminal region of the single polypeptide (Lindsley and Wang, 1991). The catalytic sites for DNA breakage and rejoining in the type II DNA topoisomerases are less well defined, although the active-site tyrosines involved in these reactions have been identified (Horowitz and Wang, 1987; Worland and Wang, 1988). How does a type II DNA topoisomerase couple its ATPase activity to its catalysis of the passage of one double-stranded DNA segment through another? The relative simplicity of the enzyme offers a unique paradigm in the study of macromolecular movements coupled to the binding and/or hydrolysis of nucleoside triphosphates. The modulation of a pair of SV8 protease-sensitive sites in the yeast enzyme by the binding of nonhydrolyzable analogs of ATP suggests that ATP binding to the enzyme causes an interdomainal allosteric conformational change (Lindsley and Wang, 1991). We show here that this conformational change can be utilized by a DNA-bound enzyme to capture a second double-stranded DNA segment and that the type II DNA topoisomerase achieves this remarkable feat by forming a circular clamp around the DNA double helix. Results DNA Binding by Yeast DNA Topoisomerase II in the Absence of AMPPNP We have utilized the retention of protein-bound DNA on fiberglass filters (Thomas et al., 1979) to monitor the binding of various forms of DNA to S. cerevisiae DNA topoisomerase II. A mixture of various forms of a 2 kb plasmid DNA was incubated with the yeast enzyme at 30% in a low-salt buffer containing 50 mM KCI, 50 mM Tris-HCI (pH 8), 1 mM EDTA, 8 mM MgCI,, 7 mM 2-mercaptoethanol, and 100 kg/ml of bovine serum albumin. The mixture was then passed through the filter, and the filter was washed suc-

Cell 834

cessiveiy with several portions of the low-salt buffer, a high-salt buffer containing 1 M NaCI, and finally a buffer containing 0.5% sodium dodecyl sulfate (SDS), as described in detail in the Experimental Procedures and in the figure legends. Figure 1A depicts the 2-D electrophoretic patterns of DNA recovered from the filtrate and from the high-salt and SDS washes of a control sample in which a total of 0.1 pmol of the plasmid DNA was incubated in the absence of yeast DNA topoisomerase II. Most of the DNA was found in the filtrate (left panel). A small portion of each form of the DNA was apparently retained nonspecifically on the filter and came off in the high-salt wash (middle panel); little DNA was detectable in the final SDS wash (right panel). When 0.1 pmol of the DNA and 0.1 pmol of the yeast enzyme were incubated and then passed through

the filter, a significant fraction of all forms of DNA was retained on the filter and came off in the high-salt wash (Figure 1 B, left and middle panels). Furthermore, byexamining the relative amounts of the various forms of the DNA in the left and middle panel of Figure 1 B, it is apparent that the enzyme binds preferentially to negatively and positively supercoiled forms of the DNA, which were preferentially depleted in the filtrate and enriched in the high-salt wash; no significant difference in the binding of the linear, nicked circular, or relaxed circular form is detectable. The preferential binding of the supercoiled forms was previously reported for Drosophila and mammalian DNA topoisomerase II (Zedriedrich and Osheroff, 1990; Pommier et al., 1989). Competition binding experiments shown in Figures 1 C and 1 D indicate that relative to a relaxed DNA, a negatively supercoiled DNA with a specific linking difference of -0.06, or a positively supercoiled DNA with a specific linking difference of 0.04, binds more strongly to yeast DNA topoisomerase II by a factor of two to three.

After Pre-Incubation with the Nonhydrolyzable P,y-lmido Analog of ATP, Yeast DNA Topoisomerase II Binds Linear DNA but Not the Circular Forms of DNA (*

*

no enzyme

plus

C 1234

enzyme

D 5678

1234

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Q

“-xcr*L cli

Ftgure Forms

1. Binding of Yeast of a 2 kb Plasmid

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- r-1 DNA

5678

Topoisomerase

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-(+I

II to the Various

Each reactron mixture contained 0 or 0.1 pmol of yeast DNA topoisomerase II and 0.1 pmol of the plasmid In 50 nl of a low-salt reaction buffer containmg 50 mM KCI, 50 mM Tris-HCI (pH 8) 1 mM EDTA, 8 mM MgCI,, 7 mM 2-mercaptoethanol, and 100 rig/ml of bovine serum albumtn. Followtng incubation at 30°C, each reaction mixture was passed through a 6 mm microftber glass filter as described in the Experimental Procedures. Successive washes were carried out with 50 ~1 portions of the low-salt buffer, three times; the low-salt buffer plus 1 M NaCI, four times; and 10 mM Trrs-HCI (pH 8) 1 mM EDTA, and 0.5% SDS. The filtrate and the high-salt and SDS washes were phenol extracted and alcohol precipitated, and each prectpttate was resuspended and used in analysis by one-dtmensional or two-dimensronal agarose gel electrophoresis, as described prevtously (Wang et al., 1983). Southern blots of the gels were probed with 32P-labeled DNA obtained by random primtng. (A) DNA only. N, nicked crrcuiar DNA; L, lmear DNA; R, toporsomers of relaxed circular DNA; -, negatively supercoiled DNA; +, posttively supercoiled DNA. (B) DNA plus yeast DNA topoisomerase II. (C) and (D) Competittve binding of yeast DNA topoisomerase II to relaxed and negatively supercooled DNA (C). or to relaxed and posittvely supercorled DNA (D). Each reaction mixture contatned 0.1 pmol of the supercorled 2 kb DNA, 0.2 pmol of the relaxed 2 kb DNA, and varying amounts of yeast DNA topoisomerase II: 0 (lanes 1 and 2), 0.05 pmol (lanes 3 and 4) 0.1 pmol (lanes 5 and 6) or 0.2 pmol (lanes 7 and 8). Following incubatron at 30°C for 20 min, each sample was frltered and washed as described, wtth the omtssion of the hrgh-salt washrng step Odd lanes, ftltrates; even lanes, SDS washes.

In striking contrast with the DNA binding results described above, pre-incubation of the yeast enzyme with the nonhydrolyzable ATP analog abolishes binding of the enzyme to any of the circular forms of DNA in the mixture, but not its binding to linear DNA. As shown in Figure 2, retention of the various forms of circular DNA on the fiberglass filter by yeast DNA topoisomerase II upon preincubation with AMPPNP is at the background level (compare the intensities of the circular forms in the middle panel of Figure 2 with the corresponding spots of the no-enzyme control sample shown in the middle panel of Figure 1A). In contrast, linear DNA is retained by the enzyme-nucleotide complex and comes off the filter in the 1 M salt wash (intense spot in the middle panel); a small amount of the retained linear DNA survived the high-salt wash and came

filtrate

I

high-salt wash

I

SDS wash

enzyme-AMPPNP Figure 2. Btnding of Various Forms of the 2 kb pHC624 DNA Topotsomerase II Pre-Incubated wrth AMPPNP

DNA to Yeast

The yeast enzyme was first incubated for 20 min at 30°C with 2 mM AMPPNP in a buffer containing 150 mM KCI, 50 mM Tris-HCI (pH E), 1 mM EDTA, 8 mM MgCI,. 7 mM 2-mercaptoethanol, and 100 uglml of bovine serum albumin. The 2 kb pHC624 DNA was then added and the mixture was diluted with the same buffer without KCI to reduce the KCI concentration to 50 mM. Incubation was continued for 20 min at 30°C, and the reaction mixture was then used in the filter-binding assay as described previously

Topoisomerase 835

II as a Protetn

Clamp

off in the SDS wash (right panel). Significantly, no topological change of the DNA rings was detected upon their incubation with preformed AMPPNP-yeast topoisomerase II complex, in this as well as other experiments not presented here.

Specific Binding of Linear DNA to AMPPNP-Yeast DNA Topoisomerase II Complex Is Not Due to Interactions between the Enzyme and DNA Termini The possibility that only linear DNA can bind to the AMPPNP-bound enzyme because of unique interactions between the ends of the DNA and the protein-nucleotide complex is supported neither by the experiment described below nor by the observation (described in the next section) that the ends of the bound linear DNA are readily joined by the NAD-dependent E. coli DNA ligase. In the experiments shown in Figure 3, we took advantage of the type II topoisomerase-mediated cleavage of double-stranded DNA (reviewed in Wang, 1985; Liu, 1989; Hsieh, 1990) to mark the positions of the DNA-bound enzyme molecules. Yeast DNA topoisomerase II was pre-

a : ctrcular

E E-AMPPNP VP16

N2-.

Ln-

DNA

b

linear

12

34

12

++ -

++ + -+

++ -

DNA

34 - ++ + -+

-0

incubated with or without AMPPNP, followed by the addition of a linear or circular DNA. AMPPNP was then added to samples pre-incubated in the absence of the nucleotide, followed by the addition of the drug VP-16 (etoposide), which enhances DNA cleavage by eukaryotic DNA topoisomerase II, to one of each pair of samples. Finally, a mixture of SDS and proteinase K was added to each sample to effect DNA cleavage by the topoisomerase and to remove the DNA-linked protein. Figure 3a shows that with a circular DNA substrate, cleavage of the DNA was observed only in the sample in which the topoisomerase was pre-incubated in the absence of AMPPNP (lane 2); DNA cleavage by the yeast enzyme pre-incubated with AMPPNP was insignificant (lane 4). With a linear DNA substrate, however, DNA cleavage was observed whether the enzyme was pre-incubated without (lane 2 of Figure 3b) or with AMPPNP (lane 4 of Figure 3b), and in either case the presence of cleavage sites far removed from the ends of the linear DNA is evident. If the binding of linear DNA to the enzyme-nucleotide complex is due to specific interactions between its termini and the enzyme-nucleotide complex, cleavage sites in the DNA should be confined to a small region near each end, and the major cleavage products are expected to be only slightly shorter than the intact linear DNA. The above experiments therefore indicate that the specificity of the enzyme-nucleotide complex for linear DNA is most likely due to a topological feature of the complex: the DNA binding and cleavage site in the complex is accessibleonly through a hole in the complex, and only linear DNA can be threaded through the eye of this protein needle.

A Topological Lock between Clamp and a DNA Ring

--

Figure 3. Binding of Yeast DNA Topoisomerase II or Its AMPPNP Complex to Crrcular and Lmear 2 kb pHC624 DNA and the Subsequent Cleavage of the DNA by the Topoisomerase in the Presence of VP-1 6 (Etoposide) Preincubation of yeast DNA topoisomerase II with or without 2 mM AMPPNP and the addition of DNA to the preincubated mixture were done as described m the legend to Figure 2. Each reaction mtxture (50 ~1) contained 0.2 pmol each of the DNA and the yeast enzyme. FoIlowIng tncubatton at 30% for 5 mtn. AMPPNP was added to 2 mM final concentration in samples that were pre-incubated In the absence of the nucleotide, and incubation was conttnued for 10 min. The reaction mtxtures were dtluted with the incubation buffer without KCI to reduce the KCI concentration to 50 mM, and VP-16 (etoposide) was added to the even-numbered samples to a final concentration of 100 ttg/ml. After 10 min at 30% SDS and proteinase K were added to each reaction mixture to a final concentration of 1% and 100 uglml, respecttvely. lncubatton was continued for 60 mtn before processing the samples for gel electrophoresis. The numbers l-4 denote the gel lanes, and plus and minus signs under each number specify the presence (+) or absence (-) of etoposide (VP16), and free yeast DNA topotsomerase II (E) or Its AMPPNP complex (E-AMPPNP) at the time of addition of DNA. N2, L2. and C2 in the left margin Indicate the positions of the nicked circular, linear, and relaxed circular form of the 2 kb plasmid, respectively.

a Circular

Protein

The idea of a hole in the AMPPNP-DNA topoisomerase II complex is further supported by the ligation of the ends of the bound linear DNA. Yeast DNA topoisomerase II was pre-incubated with AMPPNP, and a mixture of a 2 kb circular DNA and a 3 kb linear DNA was then added. Following incubation, the mixture was split into two equal portions, and E. coli DNA ligase and its cofactor NAD were added to one of the pair of samples. The use of the bacterial rather than phage T4 DNA ligase in this experiment obviates the addition of ATP to the reaction mixture, which might affect the stability of the AMPPNP-topoisomerase II complex (Tamura et al., 1992). As expected from data presented earlier, in the unligated sample only the 3 kb linear DNA was retained on the filter, most of which came off the filter in the high-salt wash (lane 2 of Figure 4) with a residual amount in the SDS wash (lane 3 of Figure 4). Upon ligation of the DNA, however, little DNA was detectable in the high-salt wash (lane 5 of Figure 4); instead, the 3 kb circular ligation product of the enzyme-bound linear DNA appeared in the SDS wash (lane 6 of Figure 4). This finding is entirely consistent with the conclusion drawn from experiments described in the sections above, i.e., the binding of AMPPNP to yeast DNA topoisomerase II induces it to form a circular clamp, and the threading of the linear DNA through the protein annulet is responsible for its preferential binding to the enzyme-

Cell 636

unligated

ligated

123

4

56

-L3

I)

mB

-c3

-C2

Frgure 4. Filter Bindmg Measurements of a Mixture of a 2 kb Relaxed Circular DNA and a 3 kb Lrnear DNA to the Yeast DNA Topoisomerase II-AMPPNP Complex Lanes l-3: the filtrate (lane 1) 1 M salt wash (lane 2) and SDS wash (lane 3) of the sample without treatment wrth E. co11 DNA Irgase. Lanes 4-6: the filtrate (lane 4), 1 M salt-wash (lane 5), and SDS-wash (lane 6) of the sample after treatment with E. coli DNA ligase and NAD. C2, relaxed 2 kb DNA; C3, relaxed 3 kb DNA; L3, linear 3 kb DNA

nucleotide complex. Therefore, cyclization of the bound linear DNA would form a catenane between the protein clamp and the DNA ring; this topological linkage between the protein and the DNA can not be disrupted by high salt, and the bound DNA is released only upon denaturation of the protein annulet by SDS. Previously, it had been observed that the addition of AMPPNP to DNA-bound Drosophila DNA topoisomerase II yielded a salt-stable complex if the DNA was in the circular form, but not if the DNA was in the linear form (Osheroff, 1986). This observation was attributed, however, to linear diffusion of the protein along the DNA. Is the Nucleotide-Modulated Protein Clamp Involved in the Capture of a DNA Double Helix for Transport through the Type II Topoisomerase-Mediated DNA Gate? The experiments described above provide strong evidence that the binding of AMPPNP to yeast DNA topoisomerase II can trigger the closure of the protein clamp to form an annulet, through which a linear DNA can access the site for DNA breakage and rejoining. To facilitate the passage of one duplex DNA segment through another, however, a pair of DNA helices must be brought together by a type II DNA topoisomerase. The preferential binding of supercoiled DNA by the enzyme in the absence of the ATP analog suggests that the enzyme can interact with two DNA segments simultaneously (see Figure 1; Zechiedrich and Osheroff, 1990). The experiments described below indicate that the clamping action of the type II enzyme is most likely involved in the capture of a DNA duplex for transport across a DNA segment already bound to the enzyme. To simplify the interpretation of the interactions between the enzyme and two DNA segments, we examined the

catenation reaction in which the two participating DNA segments reside on different molecules; reactions were also carried out in a low-salt medium so that a bound DNA would not be displaced by another DNA subsequently added. Figure 5 demonstrates the nonexchangeability of DNA bound to yeast DNA topoisomerase II in the low-salt binding buffer employed. The enzyme (0.5 pmol) was first incubated with a 376 bp DNA ring (0.4 pmol), and a 2 kb DNA ring (0.8 pmol) was then added. Following incubation, the mixture was split into two, and VP-l 6 was added to one to effect DNA cleavage by the topoisomerase. As shown in Figure 5, the small 376 bp ring was depleted from the filtrate, owing to its binding to the topoisomerase (lane l), and eluted from the filter in the SDS wash (lane 2); the 2 kb DNA subsequently added appeared only in the filtrate (lane l), and no protein-bound form was detectable in the SDS wash (lane 2). When VP-l 6 was added to the mixture to effect topoisomerase-mediated DNA cleavage, only linearization of the small ring was detectable (Figure 5, lane 3). The same experiment was repeated by inverting the order of addition of the two DNAs, and, as expected, binding and cleavage of only the 2 kb DNA added first were observed in this case (Figure 5, lanes 4-6). As shown in Figure 6, if yeast DNA topoisomerase II was first incubated with AMPPNP, subsequent incubation with

123

456

-

w-Ne -Ln

L 376 =c Frgure 5. Nonexchangeabilityof erase II in the Low-Salt Medium

376

DNA Bound to Yeast DNATopoisom-

The enzyme (0.5 pmol) was first incubated with a 0.376 kb minicrrcle (0.4 pmol) in 100 nl of the low-salt reaction mixture containing 50 mM KCI, 50 mM Tris-HCI (pH 6) 1 mM EDTA, 6 mM MgCI,. 7 mM 2-mercaptoethanol, and 100 nglml of bovine serum albumin. After 10 min at 30°C, 0.6 pmol of relaxed 2 kb DNA rings were added, and incubation was continued for 10 more min. The reaction mixture was then divided into two equal portions, one of which was used for measurement of topoisomerase II-mediated DNA cleavage rn the presence of etoposide, as described before. Lane 1, the filtrate; lane 2, SDSwash; lane 3, DNA after cleavage by the yeast enzyme in the presence of etoposide. The same experiment was carried out by inverting the order of incubation with the two DNAs; lanes 4-6 for this sample correspond to lanes 1-3, respectively, for the preceding sample. N, L, and C denote the nicked, linear, and relaxed circular forms as before, and the number following each symbol denotes the size of the DNA in kb.

Topoisomerase 837

II as a Protein

123

45

Clamp

6

Figure 6. Yeast DNA Topoisomerase II-Catalyzed Formation of a Homodimeric Catenane between Two 0.376 kb Mmicrrcles and a Heterodimeric Catenane between a 0.376 kb Minicircle and a 2 kb DNA Ring Lane 1: 0.1 pmol of the yeast enzyme and 2 mM AMPPNP were first incubated for 20 min at 30°C in a medium containing 150 mM KCI, 50 mM Tris-HCI (pH 8) 1 mM EDTA. 8 mM MgCI,, 7 mM Z-mercaptoethanol, and 100 frglml of bovine serum albumin. The minicircular 0.378 kb DNA and the larger 2 kb DNA (0.25 pmol each) were then added, and incubation was continued for 20 min before the termination of the reaction by the addition of SDS and proteinase K. Lane 2: 0.1 pmol of the yeast enzyme was first incubated for 10 mm at 30°C with 0.25 pmol of the minicircular DNA, in 50 mM KCI, 50 mM Tris-HCI (pH E), 1 mM EDTA, 8 mM MgCI,, 7 mM 2-mercaptoethanol, and 100 pg/ ml of bovine serum albumin. AMPPNP was then added to 2 mM, and, following incubation at the same temperature for 20 mm, 0.25 pmol of relaxed 2 kb DNA rings was added. The reaction was terminated by the addition of SDS and proteinase K after an additional 10 min of incubation at 30°C. Lane 3: 0.1 pmoles of the yeast enzyme was first incubated with 0.25 pmol of the minicircles for 10 min at 30%. in the 50 mM KCI low-salt buffer. Relaxed 2 kb DNA rings (0.25 pmol) were then added, and, after 10 min at 30°C, AMPPNP was added to 2 mM. The reaction was terminated 20 min later by the addition of SDS and proteinase K. Lanes 4 and 5: same as the sample shown in lane 3, except that etoposide was added to the lane 5 sample to a final concentration of 100 pglml 10 min before the termination of the reaction. Lane 6: marker DNAs used in the identification of the various species.

a mixture of the small 376 bp ring and the large 2 kb ring caused no detectable topological changes in the DNA (lane 1). If the free enzyme was first incubated with the small ring, followed by incubation with AMPPNP and, finally, the addition of the 2 kb DNA ring, a small amount of dimeric catenane of the 376 bp ring was detectable, but no catenane containing the 2 kb ring was present in the reaction mixture (Figure 6, lane 2). In contrast, when the enzyme was first incubated with the smaller DNA ring, and the larger DNA ring was then added before the final incubation with AMPPNP, the formation of a dimeric catenane between the 376 bp and the 2 kb DNA rings, as well as a dimeric catenane between two 376 bp rings, was evident (Figure 6, lane 3). In these experiments, the identity of the catenanes was established from their gel electrophoretic mobilities and from their products upon digestion with restriction enzymes that cut singly in one of the two rings; the band assigned as the dimeric catenane between the 376 bp and the 2 kb ring, for example, disappears

either by digesting the sample with Sphl, which cuts only the smaller ring, or BamHI, which cuts only the larger ring (data not shown). The above experiments can be interpreted as follows. First, as expected from the data already presented, preincubation of the topoisomerase with AMPPNP closes the protein clamp, and neither of the two DNA rings can reach the catalytic site for DNA breakage and rejoining; thus, no topological transformation of the DNA rings is possible (Figure 6, lane 1). Second, when incubation of the enzyme with the 376 bp DNA ring was followed by incubation with AMPPNP, the DNA-bound enzyme, like the DNA-free form described earlier, closes its jaws to form a DNA-bound annulet. In this state, the catalytic site of the enzyme for DNA passage is not accessible from the outside and therefore no catenane can form between the enzyme-bound 376 bp DNA ring and the 2 kb DNA ring added following the incubation of the enzyme-small DNA ring complex with AMPPNP (Figure 6, lane 2). The formation of dimeric catenane between two 376 bp rings in the lane 2 sample can be attributed to the binding of a second small DNA ring by a DNA-bound enzyme prior to the addition of AMPPNP; trapping and transport of this second ring through the bound first ring upon the addition of AMPPNP yield the homodimeric catenane. Third, when the enzyme is first incubated with the small DNA ring, the DNA occupies the site for DNA cleavage and rejoining, and, in the low-salt binding buffer employed, can not be replaced by the 2 kb DNA subsequently added; in other words, the DNA gate resides in the 376 bp DNA ring. Although the bound DNA segment containing the DNA gate can not be displaced by the subsequently added 2 kb DNA ring, the larger ring can enter the channel between the open jaws of the enzyme clamp and serve as the DNA segment for transport through the DNA gate in the smaller ring. When the open jaws close following the addition of AMPPNP, the 2 kb DNA ring is trapped and transported through the DNA gate and becomes catenated with the 376 bp DNA ring. To show that in the experiments described above the DNA gate was always present on the 376 bp ring rather than the 2 kb DNAsubsequently added, VP-1 6 was added, after the final incubation with AMPPNP, to one of a pair of samples identical to the oneshown in Figure 6, lane 3; SDS and proteinase K were then added to the pair of samples to effect DNA cleavage. As shown in lanes 4 and 5 of Figure 6, cleavage occurred only in the 376 bp ring to give the 376 bp linear form. Discussion A major finding of this work is that the binding of the nonhydrolyzable (3,y-imido analog of ATP to yeast DNA topoisomerase II triggers a conformational change of the homodimeric enzyme such that it can bind linear but not any circular form of DNA. This specificity for linear DNA is not due to interaction between the ends of the DNA with the AMPPNP-DNA topoisomerase II complex: the enzyme can cleave the bound DNA at sites far removed from the ends, and the ends ol the topoisomerase-bound DNA remain accessible to DNA ligase. Thus the specificity for

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linear DNA can be interpreted in terms of a topological and geometric constraint in the nucleotide-bound enzyme that prevents the access of any circular DNA to the DNAbinding pocket. Asshown by the DNA ligation experiment, conversion of the bound linear DNA to the circular form can be readily carried out, and the circular form remains enzyme bound. We attribute this topological and geometric constraint to the nucleotide binding-triggered formation of a hole in the type II DNA topoisomerase, with the dimension of the hole sufficiently large for a linear DNA to thread through, but too small for a duplex DNA doubled on itself to enter. That a type II DNA topoisomerase can form a circular clamp around a DNA double helix is further supported by the salt stability of circular but not linear DNA bound to the enzyme-AMPPNP complex. It makes no difference whether AMPPNP isaddedfollowing the bindingof acircular DNA to the enzyme or the nonhydrolyzable ATP analog is added to the enzyme first, followed by the binding of a linear DNA to the enzyme-nucleotide complex, and, finally, the cyclization of the bound DNA. This unique salt stability of enzyme-bound circular DNA in the presence of AMPPNP was observed several years ago (Osheroff, 1986), but the phenomenon was attributed to linear diffusion of the protein along DNA. Here we view the salt stability in terms of a topological link between the protein and the DNA ring. Recent 3-D structural dataof the 6-subunit of E. coli DNA polymerase III have provided a striking precedent of a protein ring encircling a DNA double helix (Kong et al., 1992). Formation of a topological complex between circular DNA and mammalian DNA topoisomerase II in the absence of AMPPNP was reported by Pommier et al. (1989). The nature of the complex they observed is unclear; one interpretation is that trapping of circular DNA in their experiments isdue to attachment of multiple domains of the same enzyme to the filter. The idea that topological transformations catalyzed by type II DNA topoisomerases might involve the passage of a duplex DNA through a channel between the two halves of an enzyme molecule was first raised a decade ago (Mizuuchi et al., 1980; Wang et al., 1980; Morrison et al., 1980). Based primarily on high resolution electron microscopic studies of bacterial DNA gyrase, Kirschhausen et al. (1985) proposed both that there might be a channel between the two Gyr B protomers of the holoenzyme and that a DNA segment inside the channel might be trapped and transported through the DNA gate in the enzyme when the two Gyr B polypeptides touch each other upon the binding of ATP. X-ray diffraction results of a complex between AMPPNP and a 43 kd N-terminal fragment of the E. coli Gyr B protein have also indicated the presence of a hole in the dimeric protein, and the diameter of the hole, approximately 20 A, is about the same dimension as the diameter of a DNA double helix (Wigley et al., 1991). While it is tempting to identify the hole in the crystal with the one inferred from the studies reported here, this extrapolation should be viewed with caution. First, among the 14 amino acids in the N-terminal arm of E. coli Gyr B that appear to be of key importance in forming the dimer contacts in the crystal (Wigley et al., 1991), only a few can be considered

as highly conserved among all type II DNA topoisomerases of known sequences. Second, the observed hole in the crystal structure is enclosed entirely by the N-terminal half of Gyr B, corresponding to the first 400 amino acids of yeast DNA topoisomerase II. The hole inferred from this work, on the other hand, must permit access to the site for DNA breakage and rejoining, which involves Tyr783 of yeast DNA topoisomerase II (Worland and Wang, 1989); in E. coli DNA gyrase, the corresponding active site tyrosine is Tyr122 of the Gyr A subunit (Horowitz and Wang, 1987). It is likely however, that parts of the walls lining the hole observed in the crystal of the Gyr B fragment also define the walls of the circular clamp formed by an intact type II DNA topoisomerase. How is the nucleotide binding-induced formation of a circular protein clamp related to the enzyme-mediated passage of one duplex DNA through another? It is well established that the type II DNA topoisomerase-catalyzed topological transformation of DNA involves minimally two double-stranded DNA segments: a “gate” or G segment within which a pair of staggered cuts are made through nucleophilic attacks of a pair of tyrosyl hydroxyls, and a T-segment that is to be transported through the enzymeoperated DNA gate in the G segment (for recent reviews, see Maxwell and Gellert, 1986; Hsieh, 1990). Several observations support the notion that the protein clamp is used to capture the T segment and to facilitate its transport through the G segment. First, in the present work we have deliberately chosen a low-salt medium for measurements involving the binding of yeast DNA topoisomerase II to DNA. In this medium, binding of the enzyme to the G segment is processive: a bound G segment, which can be identified by its cleavage upon the addition of a protein denaturant, can not be displaced by excess DNA in the solution (see Results). In the absence of AMPPNP, a G segment-bound enzyme can apparently interact with a second DNA segment, presumably the T segment. The preferential binding of type II DNA topoisomerase to negatively or positively supercoiled DNA, relative to binding to linear, nicked, or relaxed circular DNA, is entirely consistent with this view (Zechiedrich and Osheroff, 1990, and Figure 1). Second, as shown in the Results, the G segment binding site is accessible through the hole in the AMPPNP-bound protein clamp, but not from its exterior. This suggests that the T segment is located in the region of the hole before its crossing of the DNA gate. Furthermore, we have shown that starting with yeast DNA topoisomerase II bound to a small DNA ring, under processive binding conditions a second DNA ring can apparently be trapped by the enzyme when it clamps shut upon the binding of AMPPNP: as shown in Figure 6, a dimeric catenane between the initially bound small ring, within which the G segment lies, and the subsequently added large ring containing the T segment can be obtained under these conditions. Based on these results, a model can be constructed to explain the coupling of ATP usage and DNA duplex passage by a type II DNA topoisomerase (Figure 7). The enzyme in the presence or absence of a DNA G segment can be viewed as a molecular clamp: the clamp is open in

;Tjoisomerase

II as a Protein

Clamp

P + ATP

Frgure 7. An Illustration of a Type II DNA Toporsomerase Dependent Protern Clamp

as an ATP-

(a) In the absence of ATP (left side of drawing), the clamp is open whether the enzyme is by Itself or is bound to a DNA segment, the G segment. In thus open state, a second DNA segment can enter the molecular trap. (b) The binding of ATP to the type II enzyme closes the clamp. If a T segment is present in the trap at the time of closure, it IS caught for transportation through the enzyme-mediated DNA gate in the G segment (right side of drawing). Dimensions of the two halves of the enzyme relatrve to the DNA are drawn based on the electron microscopy results of Krrchhausen et al. (1985)for E. coli DNA gyrase, as all known type II DNA topoisomerases are evolutionarily and structurally homologous (Caron and Wang, 1992).

the absence of ATP and shut when ATP is bound to it. It is plausible that a single bound ATP per dimeric holoenzyme, rather than one bound ATP per protomer, is sufficient to close the clamp, but this is an issue to be addressed elsewhere (J. E. Lindsley and J. C. W., unpublished data). When a G segment-bound protein clamp is in the open state, there is a certain probability that a T segment can move into this Venus’s fly-trap. We believe that this probability is dependent on DNA chain statistics as well as on protein-DNA interactions: both the effective concentration of a T segment being found in the region of the hole and protein-DNA interactions would facilitate the entrance of the T segment into the trap. The binding of ATP closes the protein clamp and traps the T segment inside. Kinetic analysis to be presented elsewhere indicates that the trap may close without capturing a DNA; in other words, the coupling between ATP usage and DNA passage is not tight (J. E. Lindsley and J. C. W., unpublished data). It is likely, however, that the closing of the clamp alters interactions between the T segment and its binding site and thus facilitates its exit through the DNA gate. Therefore if a T segment has entered the molecular clamp, it will cross the G segment with a high probability when the clamp closes through nucleotide binding. In the present model, a DNA-bound type II topoisomerase utilizes ATP binding and hydrolysis to close and open a molecular trap for the capture of a second DNA double helix, which is then transported through a DNA gate in the originally bound G segment. The model extends a similar idea expressed in Kirchhausen et al. (1985) but differs from the more recent model outlined in Pommier et al. (1989) and Wigley et al. (1991). In the latter work on the 43 kd fragment of E. coli Gyr B protein, the presence of the 20 A hole in the dimeric protein has led the authors to suggest that the hole might serve to hold the T segment after, not before, its transport through the DNA gate. While the ATP-dependent closing and opening of a pro-

tein clamp provides a key piece of the puzzle as to how a type II DNA topoisomerase couples ATP usage to the transport of one DNA through another, results reported in the present study do not address the equally important question of how the T-segment exits from the protein, following its transport through the DNA gate, to complete the reaction cycle of the enzyme. Further study is needed to determine whether the post-passage T segment exits from the same opening of the clamp through which it entered (the one protein gate model), or through a second protein gate on the other side of the G segment (the two protein gate model). Experimental

Procedures

Materials Yeast DNA topoisomerase II was kindly provided by Mr. James Berger The enzyme was purified from S. cerevisiae strain JELl harboring a yeast DNA topoisomerase II overexpression clone YEpTOP2PGALl (as described in Worland and Wang [1989] and Lindsley and Wang [1991]) and stored at a concentration of 10 mg/ml at -70%. A working stock of the enzyme was kept at 50 nglml and -20% in 50 mM TrisHCI (pH 8) 1 mM EDTA. 500 mM KCI, 7 mM 2-mercaptoethanol, 100 pglml bovme serum albumin, and 50% (V/V) glycerol. A 3 kb plasmid pBluescript (Stratagene) and a 2 kb plasmid pHC624 derrved from pBR322 were used in most of the experiments. Various forms of these plasmids were obtained as follows: relaxed, by treatment with vaccinia vrrus topoisomerase (kindly provided by Dr. Ryo Hanai); linear, by treatment with EcoRl restriction endonuclease; positively supercoiled forms, by relaxation of plasmid DNA in the presence of stoichiometritally bound E. coli DNA gyrase (see Liu and Wang, [1978]; relaxation of supercoiled DNA with vaccinra topoisomerase was carried out in the presence of approximately 1 gyrase holoenzyme per 200 bp of DNA), or by supercoiling with the GAL4-T7 chimeric RNA polymerase in the presence of ribonucleoside trrphosphates and E. coli DNA topoisomerase I (Ostrander et al., 1990; following deproteination by phenol extraction, the triphosphates were removed by exhaustive dialysis against 1 M NaCI, 10 mM EDTA [pH 81, prior to alcohol precipitation of the DNA). The 376 bp relaxed mintcircle was prepared from a plasmid in which a 368 Nael segment of pBR322 was ligated with octameric EcoRl linkers and inserted into the EcoRl site of pHC624; following digestion with EcoRl and cyclizatron of the linear fragments in the presence of T4 DNA ligase and ATP, the mrnicircular DNA was gel isolated after the removal of ATP (see above). Protein-Mediated DNA Binding to Glass-Fiber Filters The method of Thomas et al. (1979) was applied in the study of interactions between yeast DNA topoisomerase II and various forms of DNA. Filters 6 mm in diameter were cut from standard size Whatman GFlC microfiber glass filters with a cork borer, and each filter was preincubated in 200 nl of 100 frglml salmon sperm DNA in a reactron buffer containing 50 mM Tris-HCI (pH 8) 1 mM EDTA, 8 mM MgC& 7 mM 2-mercaptoethanol, and 100 Kg/ml of bovine serum albumin. Standard 1 5 ml disposable Eppendorf centrifuge tubes with concave caps were used in the filtration of the samples. A preincubated filter was placed in the concave cap of a centrifuge tube, in which a small drainage hole had been pierced through the cap near the perimeter of the concavity. A reactron mixture 50 fd in volume was placed on top of the filter, and the tube was placed in an Eppendorf centrifuge with the drainage hole away from the center of rotation. The filtrate was recovered from the centrifuge tube following a brief spin of 5-l 0 s. The filter was washed successrvely wrth various 50 ~1 portrons of washing solutions as described. Acknowledgments We thank James Berger for providing yeast DNA topoisomerase II and Janet Lindsley for discussions. This work was supported by a grant from the United States Public Health Services (GM24544). The costs of publication of this article were defrayed in part by the payment of page charges. This artrcle must therefore be hereby

Cell 840

marked “advertisement” in accordance solely to indicate this fact. Recerved

August

11, 1992; revised

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18 USC Sectron

September

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