DNA repair: models for damage and mismatch recognition

Mutation Research 447 Ž2000. 49–72 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres

Mutation Research Frontiers

DNA repair: models for damage and mismatch recognition Scott R. Rajski, Brian A. Jackson, Jacqueline K. Barton

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DiÕision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, 91125, USA Received 24 August 1999; received in revised form 3 September 1999; accepted 6 September 1999

Abstract Maintaining the integrity of the genome is critical for the survival of any organism. To achieve this, many families of enzymatic repair systems which recognize and repair DNA damage have evolved. Perhaps most intriguing about the workings of these repair systems is the actual damage recognition process. What are the chemical characteristics which are common to sites of nucleic acid damage that DNA repair proteins may exploit in targeting sites? Importantly, thermodynamic and kinetic principles, as much as structural factors, make damage sites distinct from the native DNA bases, and indeed, in many cases, these are the features which are believed to be exploited by repair enzymes. Current proposals for damage recognition may not fulfill all of the demands required of enzymatic repair systems given the sheer size of many genomes, and the efficiency with which the genome is screened for damage. Here we discuss current models for how DNA damage recognition may occur and the chemical characteristics, shared by damaged DNA sites, of which repair proteins may take advantage. These include recognition based upon the thermodynamic and kinetic instabilities associated with aberrant sites. Additionally, we describe how small changes in base pair structure can alter also the unique electronic properties of the DNA base pair p-stack. Further, we describe photophysical, electrochemical, and biochemical experiments in which mismatches and other local perturbations in structure are detected using DNA-mediated charge transport. Finally, we speculate as to how this DNA electron transfer chemistry might be exploited by repair enzymes in order to scan the genome for sites of damage. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Energetic recognition; DNA repair; Base flipping; Pinch–push–pull; DNA-mediated electron transfer; Oxidative damage; Long-range charge transfer

1. Introduction Through the life of any organism, maintaining the integrity of the genetic material is of critical impor-

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Corresponding author. Tel.: q1-626-395-6075; fax: q1-626577-4976. E-mail address: [email protected] ŽJ.K. Barton..

tance. Two basic mechanisms lead to alterations in the base sequence of the DNA and thereby degrade the information content of DNA. Direct mis-incorporation of bases during genetic replication or chemical damage to the nucleic acid can occur. Although the accuracy of DNA polymerases is very high, each time the genome is copied during cell division a small percentage of bases are inserted incorrectly. As a result, in each new duplex, a number of mis-

0027-5107r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 1 9 5 - 5

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matched base pairs exist. In addition, over time nucleic acids are subject to a constant barrage of damage processes which can lead to chemical modifications to the polynucleotide. These include redox events and alkylations to yield lesions preferentially on the base moiety. In the absence of immediate cell killing, the end result of base mis-incorporation and DNA damage is the same, a mutation. Before a mutation occurs, the recognition and repair of aberrant nucleotides, formed either by misincorporation or damage, are carried out by several distinct, if not completely independent pathways: Ži. direct damage reversal ŽDDR.; Žii. base excision repair ŽBER.; Žiii. nucleotide excision repair ŽNER.; Živ. mismatch repair ŽMMR.; and Žv. recombinational repair ŽRER.. Extensive studies have focused on the chemical routes to repairing lesions and mismatches once they have been detected. NER, for example, eliminates DNA damage through a process consisting of recognition, endonucleolytic scission to either side of the damage site, and subsequent release of a 24–32 nt single-stranded piece of DNA containing the DNA lesion. The resulting sequence gap is then rapidly filled in by the action of DNA polymerase, followed by ligation back into the original substrate from which damage was removed. The BER enzymes are much smaller and often operate singly or as constituents of much smaller multi-protein complexes than those involved in NER. BER involves the selective identification and removal of the aberrant base via cleavage of its glycosidic bond; this glycosylase activity is a trademark of the two classes of BER enzymes. But how are these lesions and mismatched nucleotides first recognized? To fully appreciate the actions taken by the BER and other repair enzymes in order to protect the genome, it is important to have some grasp of the number of lesions which these repair systems must recognize and counter. Recent efforts by Ames et al. have placed the approximate number of oxidized base lesions excreted per cell per day Žpcpd. to be 11,500 for humans Ž5 = 10 13 total cells. w1x. This value is proposed to be comparable to the number of abasic sites resulting from spontaneous depurination. The oxidants responsible for this extensive damage are believed to originate as reactive oxygen species which leak from the mitochondria and endoplasmic reticulum w2,3x. Analogous estimates have been made

in rats which, using 8-oxo-dG as a bio-marker for oxidative damage and assuming that this lesion comprises 5% of all oxidative adducts, measure the total number of lesions at 24,000 pcpd w1x. This number is believed to be equivalent to or higher than estimates for endogenous non-oxidative lesions and is also comparable to the number of adducts resulting from known environmentalrdietary carcinogens w1x. If these values can be extrapolated to humans, then it stands to reason that the number of base damage events resulting from the sum of spontaneous depurination, oxidative modification, and non-oxidative base alterations is on the order of 50,000 damage events per day per cell. By contrast, polymerase-driven mistakes account for a much more modest number of errors. The human genome is composed of 3 = 10 9 base pairs and undergoes one cycle of replication per day. The polymerases reponsible for replication, on average, incorporate 1 mistake per 10 4 –10 5 bases but also possess a proofreading function which improves the fidelity of replication some 10 2 –10 3 fold w4x. Thus, a conservative estimate Žas replication is not concurrent within all cells. dictates that one error per 10 6 –10 8 bases is a result of faulty nucleotide incorporation. This level of accuracy, coupled with the size of the genome suggests that only 1000–2000 mismatches result due to replication mistakes. Clearly then, the greatest threat to the integrity of the genome Žbarring loss of DNA repair machinery function. is the intrinsic chemical reactivity of the nucleobases, not the inaccuracy of replication mechanisms. Here we describe some of the chemical issues to consider in elucidating how DNA repair proteins recognize their substrates for repair. We describe first some of the elements of recognition as well as current models that have been put forth. We then also describe efforts in our own laboratory to clarify some of the chemical principles involved in the successful targeting of aberrant sites on the DNA polymer. This includes a new approach for detecting perturbations in DNA based upon DNA-mediated electron transfer chemistry. Finally, based upon what we have learned, we present some new proposals regarding how proteins might recognize damage to DNA. These ideas are meant to provide some spark to catalyze new experiments and the development of new perspectives on DNA repair mechanisms

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2. Elements of recognition 2.1. Structural considerations The first generation of hypotheses put forward to explain the recognition of damaged and mismatched sites in nucleic acids focused on the structural details of aberrant sites and how those structural characteristics differed from either properly paired or undamaged DNA. These models highlighted the differences in molecular shape or functional group placement which might be exploited to identify, for example, a mismatch, an oxidized base, or cyclized photoproduct. Using X-ray crystallography, a growing database emerged for the many different geometries that damaged or mismatched DNAs may adopt w5– 10x. In the case of the mismatch-containing structures, crystallographic analysis indicated that even oligonucleotides with internal mispairs adopted a generally regular structure and appeared essentially the same as properly paired DNA. Similarly, the structure of an oligonucleotide containing the 8oxo-guanine lesion, as might be expected for such a small DNA modification, showed almost no structural differences from B-DNA w11x. The desire to study damaged or mispaired DNA sites in solution led to the application of NMR techniques to this structural characterization. These techniques initially made it possible to ask questions about the pairing and conformations of the mismatched base pairs and to determine how far mismatch induced perturbations propagated through the helical structure w12x. Overall, the structures determined by NMR spectroscopy agreed with the qualitative conclusion reached by X-ray crystallography: the incorporation of a base mismatch does not result in large changes in the overall conformation of a B-form DNA helix. This was found to be the case for A:G, G:G, C:A, A:A, T:T, and G:T mispaired DNA substrates w13–17x. With respect to DNA lesions, more variability in structure was encountered. Here, NMR structures, some of which included intercalation of a cross-linked drug molecule or displacement of DNA bases into solution by an opposing bulky lesion, revealed considerably more structural distortion than was observed in the models of DNA mismatch sites w18–22x.

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Thus, early structural hypotheses focused on the differences in geometry which could be identified between aberrant sites and standard DNA. The small differences which could be identified led to recognition hypotheses based on shape selective binding or direct readout of the functionalities of the aberrant sites. A proposal was made, for example, that the discrimination of mismatches might be based on deviation in the relative symmetry of the base pairs and their width Žfrom C-1X to C-1X of the nucleosides. as compared to standard base pairs w23x. This hypothesis was proposed in light of the fact that the GA mispair, which was the most symmetrical and similar in width to a proper G:C or A:T, is the least efficiently repaired base mismatch. Similarly, the recognition of the 8-oxoguanine base in DNA was proposed to rely on the different disposition of hydrogen bond donating and accepting functionalities in the grooves of the nucleic acid w24,25x. Analysis of the crystal structure of the thymine photodimer bound to the bacterial repair enzyme also focused on the structural details which might facilitate recognition; details which were highlighted to explain specificity included a kink in the bound DNA, the extrusion of one of the adenine bases opposite the T -) T, and electrostatic interactions with phosphate groups in the deformed backbone of the bound nucleic acid w26x. Although interesting, subsequent experimental results have shown that models based only on structural discrimination of mismatch and damage sites in DNA likely do not capture the full range of mechanisms applied in DNA damage recognition. Based upon structural considerations alone, the full range of mismatch recognition required is difficult to understand and clearly warrants continued attention w27,28x. Moreover, as illustrated in Table 1, models based upon structural recognition simply cannot explain the often wide substrate specificity of many DNA repair enzymes which identify and correct lesions of markedly different sizes and structures. 2.2. Thermodynamic and kinetic issues Base lesions and mismatches commonly induce thermodynamically destabilizing influences upon the DNA helix. This destabilization is easily measured through a reduction in the temperature ŽTm . at which

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Table 1 Sampling of repair proteins and representative substrate specificities

Endonuclease III MutY Human MutSa AlkA O 6-MethylguanineDNA Methyltransferase

Representative excision substrates

Reference

5,6-dihydro-dT, 5-OH-6-dU, thymine glycol, cytosine glycol, 5-OH-dC, alloxan Adenine base of G:A, 8-oxo-G:A, and A:C mismatches O 6-Me-dG, O 4-Me-dT, cisplatin 1,2-intrastrand ŽGpG.crosslink 3-Me-dA, 7-Me-dA, O 2 -Me-pyrimidines, 3-Me-dG, N-mustard ethylated dA and dG adducts O 6 -alkyl-dG, O 4 -alkyl-dT, alkylphosphotriesters

w40–42x

the two DNA strands containing the lesion denature compared to their undamaged counterpart. Furthermore, damage can lead to a kinetic destabilization of the helix, that is, one can measure an increase in the rate of base pair opening events at and around the aberrant site w29x. Extensive thermodynamic studies examining the rate of DNA sequence have described the consequences of mismatch incorporation in DNA w30–36x. Indeed, the thermodynamic consequences of mismatch incorporation are strongly dependent on the sequence context surrounding the mismatched bases. The impact on the free energy of the duplexes range from strongly destabilizing Ž2.72 kcalrmol. for one CC mismatch to strongly stabilizing Žy2.58 kcalrmol. for a GG mispair w35x. In addition, different mismatch sites were also observed to have dramatically different effects on the enthalpic and entropic components of the free energy. Similar thermodynamic characterization has been performed on oligonucleotides containing sites of DNA damage, with some surprising results. For example, the 8oxo-dG lesion, which does not appear to perturb DNA structure, destabilizes the helix by 1.3 kcalrmol w29x. Furthermore, kinetic destabilization is clearly associated with some damage sites. NMR studies have shown that for some mismatches, such as G:G, interconversion between different conformations of the mispair is fast, approx. 10 4 sy1 at 303 K w37x. In contrast, the G:A mismatch has been shown to have a conformational exchange rate of only 100 sy1 at 303 K w37x, comparable to the rates of opening for Watson–Crick base pairs. Perhaps this offers insight

w27,43,62,68–70,135x w44–46x w47–50x w28,51–53x

into why G:A mismatches are repaired less efficiently than are G:G mismatches. Modified DNA base pairs also show a propensity to adopt extra-helical conformations. The non-planar thymine glycol lesion, for example, has been shown to undergo significant motion on the NMR time scale and may be completely extra-helical w38x. In contrast, NMR studies of a DNA duplex containing the lesion 3, N 4 -etheno-2 X deoxycytidine opposite 2 X -deoxyguanine yielded results inconsistent with significant differences in the kinetics of base opening at the damaged base pair w39x. Here, too, however, recognition based simply upon the intrinsic local destabilization at an aberrant site does not appear to account for the range of mismatches and lesions that are recognized. In particular, the hierarchy of sites recognized by different repair proteins often shows little correlation with relative kinetic or thermal stabilities ŽTable 1. w40– 53x. 2.3. Base flipping DNA base flipping is a process wherein a nucleotide is rotated out of the double helix w54x. Remarkably, both the sugar and base moieties are extracted from the helix resulting in destruction of base-pairing hydrogen bonds and interruption of the base stack w55x. Most importantly, the base flip allows enzymatic access to the nucleotide. The first glimpse of an enzyme-bound, base-flipped DNA was rendered in the co-crystal structure of the methyltransferase ŽMtase. M. HhaI bound to a small DNA

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duplex w54,56x. Since then, crystallographic data has revealed that another Mtase, M. HaeIII, also base flips w57x. Additionally, extensive data suggest the generality of this means of chemically accessing the typically hindered nucleobases of duplex DNA w54x. More significantly, however, was the revelation that base flipping may also be a means by which DNA repair enzymes, specifically those belonging to the BER class of enzymes, repair damage within the genome w58–62x. This possibility was soon substantiated with the demonstration that uracil DNA-glycosylase ŽUDG. also effects base flipping of its target uracil w63–66x. Very recently, Ellenberger et al. have shown that binding of human N 3-methyladenine glycosylase ŽAAG. to a DNA duplex containing a mechanism based inhibitor results in a base flipped intermediate w67x. Perhaps the most thoroughly studied of the DNA repair enzymes which has been shown crystallographically to base flip the damage site of interest, UDG possesses a nucleotide-binding pocket into which the flipped out uracil makes very precise and specific contacts. Importantly, the UDG uracil binding pocket very closely resembles that of dUTP pyrophosphatase ŽdUTPase., an enzyme which hydrolyzes dUTP to dUMP and pyrophosphate prior to faulty polymerase incorporation into DNA w65–66x. Both enzymes have very similar features which structurally prohibit the mistaken binding of the other ubiquitous pyrimidines. The complementarity between a damaged or misincorporated nucleotide and the enzyme responsible for its repair has been thought to play a large role in DNA damage recognition w27,62x. In short, favorable and selective interactions between the damaged base and enzymatic binding pocket are believed to allow formation of the intermediate complex. However, while it is understandable how an ensemble of contacts within the pocket for the correct substrate would be beneficial, how such a search for complementarity by flipping each base individually would work on an efficient time scale is difficult to comprehend. The Mtases are believed to exploit this ‘‘trapping’’ mechanism, in part, due to their inordinately slow kinetics Ž k cat ; 0.03 miny1 . w59–60x. However, UDG increases the rate of uracil excision by some 20,000 fold Ž k cat ; 740 miny1 . over non-enzymatic processes w52a,52bx.

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Another important issue not addressed by a simple base-flipping scheme is the fact that the opposing base is not also probed. In the case of recognition of base lesions, this may be sufficient. However, as an example, the repair protein MutY recognizes not only 8-oxo-G:A mismatches but also G:A mismatches, and indeed it is the A, a natural base, that is excised, and thus presumably flipped w27,68–71x. Here the thermodynamics and kinetics associated with base flipping of aberrant sites versus properly paired sites may be important as a means of probing the interactions with the both strands. As a result, current models for damage recognition are now beginning to blend together many of the elements described thus far.

3. Current models for recognition of aberrant DNA Repair enzymes clearly may take advantage not only of structural, thermodynamic, and even kinetic features which distinguish aberrant sites a priori, but also features which enzyme binding can serve to enhance. Several models for how repair proteins recognize damage sites that exploit the different elements described, have therefore recently been put forth. 3.1. Energetic recognition Breslauer has proposed that repair proteins exploit primarily energetic differences to distinguish two damaged sites which might be otherwise structurally equivalent, energetic differences which the enzyme can probe andror enhance w71x. An illustration of this model for recognition may be displayed in the workings of the 38–42 kDa zinc-finger binding protein, XPA Žxeroderma pigmentosum group A.. The XPA protein possesses a zinc-finger domain which displays little or no selectivity, but also contained within the monomeric structure is a single-stranded DNA binding region which is proposed to infiltrate single-stranded regions caused by helix-destabilizing lesions w72x. It is this latter domain, which is suggested to identify lesion sites by their greater propensity to be single stranded, thus generating substrate specificity. This is further supplemented by interac-

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tions with other single-stranded binding proteins and helicases which take part in localized binding and ultimately facilitate duplex unwinding and subsequent catalysis. As pointed out by Breslauer, this mechanism, where aberrant sites in DNA are identified by the effect they have on the stability of the DNA structure, may be utilized more generally. By recognizing the effect of the lesion on the DNA rather than the structures of the lesions themselves, a protein could recognize a broad range of errant DNA structures, ranging from DNA photoproducts w73x, aflatoxin adducts, cisplatin adducts, dimerized pyrimidines, to oxidative damage and GG mismatches. 3.2. Enzyme-dependent base flipping Verdine and Bruner have offered two possible mechanisms, somewhat related to the energetics by which repair proteins locate sites of damage; both rely upon extensive enzyme-facilitated base flipping events w73x. Mechanisms involving base flipping have become increasingly favored given the increasing number of base flipping enzymes which have been characterized w27x. Crystallographic studies supplementary to those of M. HhaI, M. HaeIII, UDG and human 3-methyladenine DNA glycosylase ŽAAG. have shown that another key DNA repair enzyme, T4 Endonuclease V Ža DNA glycosylaserAP lyase that initiates repair of cis–syn cyclobutane pyrimidine dimers in DNA., also base flips w54x. T4 Endo represents a unique case however since it does not flip the damage site, but rather a nucleotide complementary to the aberrant site w54x. This readily allows access of critical enzymatic residues to the site of damage and subsequent catalytic function. Additionally, some data have suggested base flipping mechanisms for the amino-methyltransferases M.TaqI and M. PÕuII, Escherichia coli DNA Photolyase, E. coli Endonuclease III E. coli Exonuclease III, T4 b-Glucosyltransferase, E. coli Ada O6-Methylguanine DNA methyltransferase, E. coli mismatch-specific uracil DNA glycosylase, T7 ATP-Dependent DNA ligase and T7 DNA polymerase w54x. Given the apparent prevalence of base flipping proteins, a format for enzyme substrate selection based partly upon chemical complementarity Ži.e., induced fit. of the lesion within an extrahelical ac-

tive site has been put forth. Verdine and Bruner have suggested that this may occur in either processive or nonprocessive manifolds. In the processive mode, a random base is targeted for extrusion from the helix and the enzyme then migrates along the DNA duplex so as to continually exchange one extrahelical base for the next. Arrival at a lesion that displays enhanced affinity for the enzymatic binding pocket leads to a stalling of the enzyme and subsequent catalysis so as to excise the damaged moiety. Alternatively, a nonprocessive model invokes the random extrusion of bases from the duplex substrate and attempted fitting into the binding pocket. Those bases that fit lead to catalysis whereas those that do not are released and allowed to reanneal within the duplex. Importantly, energetic and probability considerations suggest that a nonprocessive mechanism would be exceedingly inefficient. Thus, the more likely manifold is that entailing the processive migration of the enzyme and its extrahelical companion. Although this might seem energetically feasible Žmigration of an already installed extrahelical site would be anticipated to proceed with little, if any, additional energetic investment., such a mechanism, involving the extrusion of each base, would seem to be quite slow. In fact, such a scanning motif has not yet been visualized. 3.3. Pinch–push–pull But would complete extrusion be required? As suggested above, this would be necessary in order to rigorously check the complementarity between enzyme and lesion. Could ‘‘induced fit’’ be achieved via some other means? Probably not, but perhaps more intriguing to contemplate is the recent ‘‘pinch–push–pull’’ mechanism of scanning suggested by Tainer w62x. Crystal structures of human UDG complexed with two different DNA substrates, one bearing a U:A base pair and one bearing a U:G mismatch reveal that both complexes place the extrahelical uracil within an enzyme active site pocket. What is particularly interesting is that conserved enzyme loops approach the DNA phosphodiester backbone and make contacts so as to compress phosphate-to-phosphate distances on either side of the ˚ This results in a kinkuracil by approximately 4 A. ing of the DNA by about 458 Žpinch. which gives

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rise to pushing out of the uracil due to insertion of a critical leucine residue within the base stack Žbelieved to effectively push the uracil into an extrahelical conformation.. The ‘‘pull’’ component of the mechanism is provided by the high degree of complementarity between the flipped uracil and the binding pocket of the enzyme. Quite significantly, a similar observation has been made with respect to the E. coli mismatched-uracil DNA glycosylase ŽMUG. w74x. Although the phosphodiester bond 3X of the mismatched guanosine is cut, the DNA in the co-crystal structure is clearly kinked with the uracil bound extrahelically into an active site pocket; the cavity created is filled once again by insertion of a leucine side chain. Thus pivotal to the ‘‘pinch– push–pull’’ mechanism of scanning and recognition is phosphodiester backbone compression w27,62x. Clearly then, it would appear that a processive scanning mechanism may not necessarily require the generation of a base-flipped nucleotide at each step along the way. Rather, scanning may simply be performed so as to drag a specific set of enzymephosphate contacts along the helix in a fashion so as to transiently compress the backbone. Those sites which display compromised stability within the DNA duplex relative to the native base pairs may then give rise to an extrahelical intermediate which then is captured via highly favorable interactions with the ‘‘lesion-binding’’ pocket. Alternatively, processive backbone compression may allow rapid scanning until a site of inherent duplex flexibility is encountered, at which point enzyme residency time is increased and the enzyme much more aggressively induces lesionrbase extrahelicity, perhaps using a push–pull motif as described by Tainer. Important to note at this juncture are findings by Roberts et al. pertaining to M. HhaI. In studies to address binding of M. HhaI to G:A, G:U, and G:AP ŽAP s abasic site. mismatches at the target site, it was noted that it is the DNA backbone which is targeted for rotationrcompression by M. HhaI and that the identity of the extrahelical base does not have a large influence upon the extent of binding Žwithin the series of mismatches. w55x. That the identity of the base to be flipped is not important for recognition but rather plays a role in catalysis is suggested not only for M. HhaI, but also for E. coli AP endonucleases, E. coli exonuclease III, and en-

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donuclease IIV w55x. This would seem to suggest that, at least for some repair enzymes, backbone compression plays the primary role in damage recognition. Quite importantly, such a scanning manifold provides a mechanism by which to assay the integrity of the base pair and not simply that component which could be envisioned to be encapsulated within the enzyme-lesion binding pocket.

4. Chemical model systems for the recognition of aberrant sites in DNA Our laboratory has focused on the design and application of small metal complexes to probe DNA. As part of that effort, we have developed model systems to explore how to detect aberrant sites in DNA. By constructing well defined chemical assemblies, we can examine some of the factors which may be important in targeting these sites preferentially. Here we describe two very different approaches. The first involves the design of a small complex which binds DNA mismatches and exploits the local destabilization associated with mismatched sites. The second takes advantage of a distinctly different characteristic associated with mismatched sites, their effect on the electronic structure of DNA. In this application, using DNA mediated electron transfer chemistry, mismatches and other aberrations which locally perturb the stacking of DNA can be probed with high sensitivity, and from a distance. 4.1. [Rh(bpy)2 (chrysi)]3 q a designed synthetic complex which recognizes mismatches In our research group, octahedral complexes of rhodiumŽIII. which bind DNA by intercalation have been extensively explored in order to design complexes which recognize DNA sites with high specificity w75x. These coordinatively saturated, substitutionally inert complexes have been shown to bind to DNA by the insertion of a single phenanthrenequinone diimine Žphi. ligand into the base stack. This intercalative interaction, which provides a high binding affinity for the nucleic acid, has been used to deliver recognition elements which are placed on the remaining, ancillary ligands of the complex.

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Fig. 1. Image of D-a-wRhwŽ R, R .-Me 2 trienxphix 3q, an octahedral X X rhodium complex specifically designed to bind 5 -TGCA-3 sites within canonical B-form DNA. The two views of space-filling models of the complex Žblack. bound to DNA show the high degree of chemical complementarity between the DNA binding site sequence metal complex. In addition to the intercalation of the phenanthrenequinonediimine Žphi. ligand within the base stack, binding of the complex benefits from contacts of the ancillary X X ligand methyl groups and axial ammines with 5 -TGCA-3 as shown in the bottom right-hand schematic w77,78x.

Fig. 1 shows as an example the complex D-awRhwŽ R, R .-Me 2 trienxphix 3q, which was designed specifically to bind to the site 5X-TGCA-3X w76x. With the complex intercalated in the major groove side of the DNA duplex, and oriented on the helix by this intercalation, an ensemble of non-covalent contacts between the ancillary Me 2 trien ligand and the DNA base pairs become available. In particular, hydrogen bonding contacts between the axial amines of the complex and the O 6 of guanine arise as well as van der Waals contacts between the pendant methyl groups on the metal complex and the methyl groups on the flanking thymines. A high resolution NMR structure w77x and, most recently, the crystal structure of the rhodium intercalator bound specifically to the

central 5X-TGCA-3X site of an eight base pair DNA oligonucleotide w78x both serve to confirm that this complex targets specifically the sequence 5X-TGCA-3X from the major groove, and, remarkably, with each of the predicted contacts. Using this general design strategy, metallointercalators have now been constructed which bind their target sites on DNA using shape selection, direct readout, and a combination of these. Another feature of these metal complexes which is exploited in optimizing designs is their rich photochemistry w79,80x. Upon photoactivation, the complexes cleave the sugar–phosphate backbone of DNA, thereby marking their sites of binding. Complexes have been obtained with binding affinities ranging from 10 6 –10 8 My1 , and indeed with specificities which allow their application in the site selective inhibition of DNA-binding proteins w81,82x. In the effort to design a small molecule which would bind preferentially to mismatch sites, we chose to exploit the kinetic and thermodynamic destabilization of the DNA helix associated with mismatched sites. Molecular modeling indicated that this intercalating chrysi ligand, which is wider than the phi ligand, would show significant steric interactions so as to preclude binding within a ‘‘standard’’ intercalation site. As a result, the sterically bulky DNA intercalator, 5,6-chrysenequinone diimine Žchrysi., was designed and incorporated within the bisbipyridyl rhodiumŽIII. complex illustrated in Fig. 2 w83,84x. Binding of the complex wRhŽbpy. 2 chrysix 3q to properly paired DNA would then require a significant energetic cost to disrupt the local structure of the helix. On the other hand, if base mismatches were present, the perturbation of the helical structure at and around the mismatch sites might allow more facile binding of the complex. Here then, we intended to achieve specificity not by taking advantage of structural contacts in the groove of DNA, as with earlier phi complexes, but by exploiting the thermodynamic, kinetic and indeed structural constraints within the base pair stack at a mismatched site. This approach resembles one suggested for DNA repair proteins, that of energetic recognition. In both cases, the DNA binding molecule preferentially occupies binding sites where the energetic cost of DNA distortion required for their binding has already been paid either partly or in full.

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Fig. 2. Comparison of steric width of tricyclic phi and tetracyclic chrysene ligands reveals that the chrysene ligand would demand ˚ of space upon intercalation into DNA. As shown an additional 2 A in the space-filling model below the phi ligand, just enough room is afforded in normal DNA to allow phi intercalation . Thus, intercalation of the chrysene ligand preferentially targets DNA sites which are, in some way distorted, or more easily distorted than normally base-paired sequences; simply put, the chrysene ligand is too sterically demanding for intercalation into normal DNA.

Experimentally we were able to demonstrate that DNA base mismatches can be effectively recognized using this simple steric-exclusion strategy w84x. The sterically bulky chrysene intercalator was found to bind and, upon photoactivation, cleave in the vicinity of mismatch sites and with little detectable interaction with Watson–Crick B-form intercalation sites w84x. Cleavage was seen preferentially at C:C, C:A, T:T, A:A, and T:C mismatch sites. The thermodynamic binding constant at the C:C mismatch was found to be ; 50 times greater than the non-specific binding constant. The recognition properties of the complex were then examined at the eight possible base mismatches in each of their sixteen possible single base pair sequence contexts; cleavage of the DNA at or near the mispair was observed at 82% of all possible mismatch sites w85x. In addition to this broad spectrum of activity, the specificity of the wRhŽbpy. 2 chrysix 3q for a C:C mismatch was sufficient to allow the discrimination of a single mismatch site within a 2725 base pair plasmid DNA heteroduplex. This approach therefore effectively achieves the specific targeting of a base mismatch w85x.

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How does binding by the chrysi complex compare to the analogous phi intercalator? Even if they are not as sterically bulky as the chrysene modified complexes, all intercalating complexes must first pay an energetic price to disrupt base pairing and insert themselves into the DNA helix. As a result, it would be expected that any intercalator would bind preferentially at mismatch sites since preexisting disruption in base pairing effectively lowers the energetic cost of binding. This is indeed the case. Cleavage by the phi complex is observed at the mismatch site; however, this cleavage is seen in addition to cleavage at the many other sites observed in the absence of the base mismatch. In contrast, at similar concentrations, the chrysi complex displays cleavage only at the mismatch site. Although there is an energetic benefit to an intercalator binding at an already disrupted site, this effect is not large enough to generate true specificity. DNA photocleavage intensities and the derived association constants of the chrysi complex at various mismatch sites were compared with measured values for the thermodynamic destabilization of each mismatch. Although increases in the binding constant of wRhŽbpy. 2 Žchrysi.x3q and photocleavage were generally observed with increasing helix destabilization, the correlation between the two variables demonstrated some flexibility. Such a finding is not unexpected and underscores the difficulty in assuming that measurements of thermodynamic destabilization fully represent all the effects of mismatch or lesion incorporation into the DNA helix. What wRhŽbpy. 2 Žchrysi.x 3q likely exploits is the conformational flexibility of the mismatched site; what the complex requires for binding is a site which is opened enough to allow ready access of the tetracyclic chrysi ligand. While this parameter is likely related to the thermodynamic destabilization, one cannot assume that such a thermodynamic measurement faithfully represents it. The hierarchy of recognition for this compound is depicted in Table 2. In a general sense pyr:pyr mismatches appear to be the preferred sites of damage by wRhŽbpy. 2 chrysix 3q. Interestingly, this is analogous to the general trend of selectivity observed for the repair enzyme MutS w86x. Both MutS and wRhŽbpy. 2 chrysix 3q share recognition preferences wherein pyr:pyr mismatches are recognized with high

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Table 2 Comparisons of mismatch affinity and specificity of D-wRhŽbpy. 2 chrysix 3q with various repair proteins D-wRhŽbpy. 2 chrysix 3q hMSH2 Taq HB8 MutS

D-wRhŽbpy. 2 chrysix 3q MutY ŽG:A mismatch. E. coli MutS

Specificity

Reference

CC f CT ) AC ) TT 4 AA f GA f GG f GT GT ) GG f GA ) AC G CT f TT 4 AA f CC TT G CT ) CC f GT f GG ) GA f AC ) AA

w84,85x w86x w87x

Affinity

Reference

K S : 1 = 10 7 y 2 = 10 5 My1 , K NS : 2 = 10 4 My1 K S : 3 = 10 7 My1 , K NS : 4 = 10 6 My1 K S : 3 = 10 7 y 2 = 10 6 My1

w85x w68x w88x

efficiency. In the case of the hMSH2rhMSH3r hMSH6 system w87x, the C:C mismatches, those most destabilizing to duplex DNA, are among the least efficiently repaired and in fact the trend appears opposite to that of the rhodium complex. Analysis of numerous repair enzymes has often shown that specific binding to damage-containing substrates affords a K a that is only improved by one to two orders of magnitude over the non-specific K a . This is certainly the case with MutY in the presence of a G:A mismatch versus simple native DNA w68x. Likewise, a similar trend is observed with wRhŽbpy. 2 chrysix 3q; the non-specific binding constant Ž K NS . is low but upon incorporation of a mismatch, the binding constant Žnow K S . is increased anywhere from 1 to 3 orders of magnitude. Under optimum conditions the metal complex binds with comparable efficiency to that observed with E. coli MutS protein w88x. Thus it is clear that this relatively simple small-molecule strategy can indeed allow the preferential targeting of mismatched sites Žwith often very high affinity.. Perhaps more important is that such targeting probably exploits some of the very same principles of recognition inherent to DNA repair proteins. We are currently exploring whether such complexes might also target a variety of DNA lesions with some specificity. This strategy may hold promise in the development of diagnostic and perhaps novel chemotherapeutics. Moreover, this targeting approach reveals important fundamental elements which may be important in damage recognition within the genome. Clearly, however, the factors which determine wRhŽbpy. 2 chrysix 3q specificity do not represent the whole story. Other characteristics of aberrant

sites in DNA must in fact need to be exploited by DNA repair proteins. 4.2. DNA electron transfer: detection based upon the electronic structure of DNA A completely different approach to targeting or sensing mismatches takes advantage of the distinctive electronic properties of DNA. Double helical DNA is a unique polymeric structure in solution; in its interior resides a well-ordered p-stacked array of heterocyclic bases. Indeed, it is the stacking of bases on top of one another that largely stabilizes the macromolecular structure. Solid state materials which contain analogous p-stacked arrays tend to conduct along the stacking axis w89–91x. Thus it has been suggested and debated, essentially since the double helical structure of DNA was first proposed, that the DNA base pair stack might similarly be conductive, or as described in molecular terms, that the DNA p-stack might facilitate long range charge transport w92–94x. What has become clear in the past few years, through a mix of biological, chemical, and physical experiments, is that long range charge transfer reactions mediated by the DNA helix are facile. These p-stack-mediated reactions can occur with extremely fast rates Ž5 ps. w95x, over long molecular ˚ . w96x, but, perhaps most impordistances Ž200 A tantly, these reactions are exquisitely sensitive to DNA base stacking. That long-range charge transport through DNA serves any biological function has yet to be shown. What is clear, however, is that DNAmediated electron transfer chemistry provides a sensitive probe of base pair stacking and its local perturbations.

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4.3. Spectroscopic studies of DNA-mediated electron transfer Just how facile such processes may occur was first suggested in a series of photophysical experiments where photoinduced luminescence quenching reactions of metallointercalators bound to DNA were conducted w92–94,97x. Early studies revealed that luminescence quenching rates of intercalated but non-covalently bound wRuŽphen. 2 dppzx 2q Ždppz s dipyridophenazine. were strongly dependent upon the differing binding modes of the electron acceptor and quencher, with DNA. With wRuŽNH 3 . 6 x 3q, which binds in the groove of DNA, luminescence quenching rates were consistent with a diffusion-controlled process. In contrast, in the presence of the DNA intercalating complex wRhŽphi. 2 phenx 3q, quenching occurred on a time scale too fast for diffusion; instead it was proposed that the electron transfer occurred through the base stack, given the coupling of both the intercalated donor and acceptor within the helix. In parallel studies, the two metallointercalators were tethered to either ends of a 15-mer DNA duplex and the luminescence was fully quenched, again consistent with facile long range electron transfer through the DNA helix w98x. Recent studies of photoinduced electron transfer between modified bases in DNA highlight the sensitivity of long range electron transfer to how the donor and acceptor are stacked in the helix w99x. Photooxidative quenching of two fluorescent derivatives of adenine, 2-aminopurine Ž2-AP. and N 6ethenoadenine, ´dA, by guanine was examined in DNA duplexes in which the distance between guanine and adenine derivative was systematically varied. While these modified adenines are energetically and electronically quite close, their interactions with the DNA base stack differ considerably. Owing to its steric bulk, ´dA is poorly stacked within the DNA helix, albeit associated in an intrahelical conformation w100x. In contrast, 2-AP is well stacked in DNA and able to hydrogen bond to the complementary T w101x. Because of these differing interactions within the helix, strikingly different electron transfer kinetics are observed in duplexes incorporating these modified adenines. In the case of the poorly stacked ´dA, slow electron transfer and a steep dependence of electron transfer with distance is obtained; here,

59

DNA is a poor conduit for charge transport. However, for 2-AP, which is well-coupled into the base pair stack, ultrafast kinetics are found and a very shallow dependence of electron transfer with distance is obtained. Hence efficient charge transfer through DNA relies sensitively upon how the redox partners are coupled into the DNA base pair stack. Importantly, DNA-mediated electron transfer is sensitive not only to the stacking of donor and acceptor but also to stacking of the intervening base pairs. It is in this context that the utility of DNA electron transfer chemistry in sensing mismatches was first demonstrated ŽFig. 3.. Early spectroscopic studies addressing this issue involved measurements of fluorescence quenching in DNA duplexes cova-

Fig. 3. As demonstrated in studies of electron transfer in duplex DNA, ethidium Žshown as the intercalated oval on left side of X duplex. and the rhodium complex wRhŽphi. 2 bpy x 3q represent an ideal redox couple by which to investigate long-range charge migration. It is noteworthy that incorporation of a C:A mismatch at the sequence intervening these two redox partners very significantly inhibited electron transfer as based on the results of fluorescence quenching experiments w102x.

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lently modified with ethidium ŽEt., a classical organic intercalator, and wRhŽphi. 2 bpyx3q w102x. Conjugation of each molecule to the 5X terminus of each strand within DNA duplexes ranging in length from 10–14 bp, and fluorescence quenching studies revealed significant quenching and a shallow dependence of the quenching yield on distance separating the two intercalators. But perhaps more interesting was the observation that this long range quenching could be attenuated by the presence of an intervening mismatch. By substituting the T Žbold face. of the native strand 5X-Et-CTATCTATCGT-3X to either a cytosine or guanine, mismatch-containing duplexes were generated. Hybridization of these strands to the mutual complement 5X-Rh-ACGATAGATAG-3X thus afforded three duplexes containing centrally-located T:A, C:A, or G:A pairings. C:A mismatches are known to cause local disruptions in the DNA base stack w5,6,12,15x, although the bases remain intrahelical whereas G:A mismatches are known to be of comparable stability to a normal G:C base pair w7,9,13x. While electron transfer quenching was comparable in the tethered duplexes containing well matched DNA and that with the intervening G:A pair, in the case of the intervening C:A mismatch, electron transfer quenching was dramatically diminished ŽFig. 3.. This experiment established the path for electron transfer as through the base pair stack. Furthermore this experiment demonstrated how the electron structure of DNA could be exploited in sensing base mismatches. A subsequent study exploiting the fluorescent properties of Et, but instead using 7-deazaguanine ŽDZ. as the electron donor further demonstrated the importance of the p-stack integrity upon long-range charge transfer w103x. Mispairing of DZ with A, T, or G was found to significantly diminish the yield of Et fluorescence quenching. Consistently, then, mismatches can be spectroscopically detected via longrange charge transfer chemistry. 4.4. Electrochemical approaches We have been interested in exploiting DNA electron transfer chemistry more generally in the design and development of new diagnostic assays for mismatch detection. Current technology for mismatch

detection, either using fluorescence or electrochemical assays, has predominantly relied upon the differential thermodynamic stabilization of matched versus mismatched sites w104–109x. Thus, directly or indirectly, these methods depend upon hybridization strategies. Exploiting DNA electron transfer chemistry provides a completely orthogonal and therefore hopefully a complementary approach. DNA-modified electrodes were constructed by the covalent attachment of DNA duplexes containing alkyl thiol linkers to a gold surface w110,111x. In studies of daunomycin ŽDM., a redox-active antitumor agent and intercalator, covalently cross-linked at specific guanine sites within duplex DNA, efficient long range electron transfer was observed over dis˚ w112x. The site of intercalation was tances up to 35 A controlled by incorporating a single GC base step in otherwise A–T or inosineŽI. –C sequences, since daunomycin requires the N-2 of guanine for covalent crosslinking w113x. Remarkably, efficient reduction of DM was observed regardless of its position along the 15-base-pair sequence. Not only were the intensities of the daunomycin cyclic voltametric signals the same for each DM-modified assembly, but the characteristic splittings between cathodic and anodic waves as a function of scan rate remained constant throughout the entire series. Thus, within the resolution afforded by this electrochemical method, increasing the distance between redox partners did not substantially change the rate of electron-transfer. In order to establish that the path for electron transfer was indeed through the base pair stack, and not short circuited in some way on the electrode surface, an intervening C:A mismatch was incorporated into the DNA films. Quite dramatically, this one-base change switched off the electrochemical response entirely. As a control, sequences in which the positions of the daunomycin and C:A mismatch were reversed Žsuch that the mismatch was located above the daunomycin relative to the gold. showed no decrease in electrochemical response, thus disproving possible lateral charge migration between immobilized duplexes. This general electrochemical approach to mismatch detection has been pursued in assays on DNA films using non-covalently bound intercalators as well w114x. The response of intercalating probes associated with DNA films effectively reports the pres-

S.R. Rajski et al.r Mutation Research 447 (2000) 49–72

ence of a wide variety of mismatches. Moreover, single-base changes in sequences of varied base composition are detected with high sensitivity, demonstrating the advantage of this detection approach over hybridization-based methods. Reversible in situ hybridization of oligonucleotides to probe mismatch incorporation has also been performed wherein the chemical readout is expressed as charge transfer dependent signals. The success of this dual hybridizationrelectron transfer methodology represents an important observation from the standpoint of practical assay development. For increased sensitivity, these assays have been coupled to electrocatalytic schemes w94x. This electrocatalysis greatly enhances the differentiation of complementary from mismatched duplexes, and allows the facile detection of point mutations in DNA oligonucleotides. Clearly then, even subtle disturbances in DNA structure can be readily detected via charge transfer pathways which rely intimately upon the integrity of the pstack. 4.5. Consequences of long-range charge transfer in DNA: chemistry at a distance DNA can not only behave as an efficient conduit for long-range charge transfer, but may also take part in oxidative transformations as a result of DNA mediated charge transfer chemistry w92–94x. Radiation biologists had debated whether radicals migrating through DNA might lead to ‘‘damage at a distance’’. Indeed, the long range oxidation of 5X-GG-3X sites in DNA was first demonstrated in DNA assemblies containing the tethered rhodium intercalator wRhŽphi. 2 bpyx 3q, and illustrated the formation of such oxidative damage from a distance ŽFig. 4. w115x. In parallel studies, the repair of a DNA lesion was also demonstrated w116x. In DNA assemblies containing a central thymine dimer lesion, the rhodium intercalator, tethered near the end of the DNA duplex, triggered the oxidative repair of the thymine dimer using visible light w116x. Thus the damage and also repair of DNA lesions can be promoted from a remote site along the DNA helix. The photochemistry of the RhŽIII. intercalator bound to DNA yields base photooxidation upon irradiation at low energy Ž365 nm. w92–94,115–117x, whereas irradiation at high energy Ž313 nm.

61

w75,79,80x leads to direct H-atom abstraction chemistry, affording direct strand scission. These direct strand cuts mark the sites of rhodium binding and thus verify the spatial separation of the intercalator from sites of oxidative reaction. Since these first chemical studies, other potent oxidants have been seen to promote oxidative damage to DNA from a distance, but they do not display the differential photoreactivities available with wRhŽphi. 2 bpyx 3q w96,118x. That 5X-GG-3X sites in DNA are easily damaged is a direct function of their oxidation potential w119x. The guanine base is that which is most easily oxidized, but notably, oxidative damage to G doublets proceeds with a high degree of specificity for the 5X-G. This 5X-GG-3X reactivity has really become a hallmark for damage to DNA via electron rather than oxygen transfer chemistry w120x. Extensive modeling studies have shown that within 5X-GG-3X dinucleotides, the vast majority of the highest occupied molecular orbital resides on the 5X-, and not the 3X-G w119x. The oxidation potential of a remote guanosine within duplex DNA is 1.29 V Žvs. NHE. whereas the potential of the 5X-G within 5X-GG-3X is suggested to be reduced by ; 300 mV w118–121x. Studies using tethered oxidants to examine damage at long range have shown the same characteristic reactivity as seen in spectroscopic studies of long range charge transport. Reactions appear to be relatively insensitive to the distance separating the oxidant from the damage site but are sensitive to the intervening stacking and sequence. In many of these studies, assemblies were constructed containing both proximal and distal 5X-GG-3X doublets so as to assay the sequence and structure intervening. In a series of Rh-DNA assemblies in which the intervening DNA sequence was systematically lengthened, a very shallow distance dependence in the distalrproximal damage ratio was observed over the range of 41–71 ˚ between 5X-GG-3X sites w96x. In fact, thus far, it has A been shown that damage can occur over distances of ˚ w96x. approximately 200 A Importantly, long range oxidative damage in DNA is sensitive to perturbations in the intervening polymer, DNA bulges which do not stack well into the helix such as the 5X-ATA-3X bulge, have been shown to substantially diminish damage at the distal 5X-GG3X relative to that damage observed at the proximal

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X

X

Fig. 4. Construction of DNA-Rh assemblies with multiple 5 -GG-3 sites affords us a methodology by which to examine long-range oxidative DNA damage and to examine the influence of intervening sequence and structure upon long distance charge migration. Studies X X involving the incorporation of a methyltransferase binding site ŽM. HhaI. within the sequence intervening two 5 -GG-3 revealed that X X X X significant 5 -guanosine damage occurred at both 5 -GG-3 sites; the ratio of damage is represented as the hollowed arrows above each 5 -G, the larger the arrow, the greater the extent of charge-transfer induced damage. Upon binding of M. HhaI to the Rh-DNA assembly Žand X subsequent base flipping. significant inhibition of distal 5 -G damage is observed. This demonstrates for the first time, that long-range charge transfer through DNA can be attenuated by DNA binding proteins, particularly those that are capable of extracting a DNA base from the interior of the helix w128x.

5X-GG-3X w122x. Long-range damage is also sensitive to intervening sequence, in particular intervening 5X-TA-3X steps w96x. Assemblies were constructed to compare the influence of 5X-TATA-3X versus 5XTA AA-3X insertion into the sequence intervening two 5X-GG-3X sites. Remarkably, this simple change afforded a ) 25% decrease in distal: proximal damage ratios. In contrast, charge transfer through the TAAA-containing substrate gave rise to nearly identical distal:proximal damage ratios to those observed with a random sequence of mixed GC:AT content. Rather than the effect of simple sequence composition w118x, we have attributed this sequence-dependent diminution in distal oxidation to the structural

flexibility associated with 5X-TA-3X steps. This is strongly supported by the intrinsic flexibility and poor base–base overlap, seen by crystallography and NMR, at 5X-TA-3X steps w123x. Interestingly, mismatch detection based upon DNA charge transport might be not only based upon perturbations in stacking but also based upon the differing redox characteristics associated with DNA lesions and mismatches. Notably, studies of DNAmediated charge transfer in duplexes selectively functionalized with wRuŽphen.ŽMe 2 dppz.Žbpy.x 3q ŽMe 2 dppz s 9, 10-dimethyl-dipyridophenazine. have shown that the presence of a G:A mismatch can be differentiated from that of a G:T mismatch based

S.R. Rajski et al.r Mutation Research 447 (2000) 49–72

upon oxidation potential w124x. While the more disruptive G:T pair was found to significantly inhibit long range damage to a 5X-GG-3X site distal from the site of metal–DNA intercalation, the structurally more subtle G:A mispair exhibited only slight influence upon the same 5X-GG-3X damage. Interestingly, and perhaps more important from a DNA damage standpoint was the observation of oxidative damage at the guanine involved in G:A mispairing. Similarly, extensive long-range G-damage has been observed in duplexes containing guanosine opposite a 4-methylindole modified nucleotide w125x. Whether this succeptibility to damage is the result of redox potential differences andror increased solvent accessibility has not yet been rigorously established. Steric accessibility of the mismatched G within the minor groove has been suggested by Thorp and co-workers based on similar observations using the nonintercalating metal complex wRuŽbpy. 3 x 3q w126,127x. 4.6. Protein modulation If long range oxidative damage to DNA depends sensitively upon DNA structure and sequence, can it be modulated by proteins which bind to DNA? Efforts in our laboratories to examine charge transfer modulation with a base-flipping protein involved in cytosine methylation, the methylase M. HhaI, have illuminated this possibility. M. HhaI recognizes and binds the sequence 5X-GCGC-3X . Enroute to methylation of each internal cytosine Žat the C-5 position., a base flipped complex is formed with each target cytosine in a transient extrahelical conformation; a glutamine side chain from the enzyme fills the space of the cytosine, leaving the overall structure of the DNA without significant distortions w54–56x. Utilizing the strategy described earlier, we constructed two wRhŽphi. 2 bpyx 3q-DNA assemblies which placed an M. HhaI binding site within the sequence intervening two 5X-GG-3X doublets w128x. Since a mismatch at one of the target cytosines appears to favor DNA-enzyme complexation, we examined a Rh-DNA assembly containing the sequence 5X-GCGC-3X and one containing a C U transversion at the bold-faced C w54–56x. Not surprisingly, M. HhaI binding to either duplex profoundly inhibited long-distance charge transfer, as revealed by diminution of 5X-GG-3X damage at the

´

63

site distal from points of Rh-DNA contact. Base flipping by M. HhaI clearly attenuated electron transfer through both DNA duplexes. Interesting as well was the observation of intense charge transfer-dependent damage with the DNA-protein binding site of the duplex containing the G:U mismatch. Given the comparable changes in distal:proximal 5X-GG-3X damage ratios observed for both Rh-DNA assemblies, we believe the damage within the binding site 5X-GUGC-3X might occur on a significantly different Žpresumably slower. time scale from that of the 5X-GG-3X damage events. Such processes have been observed before w129x. Utilizing Rh-DNA assemblies containing a T -) T dimer in addition to 5X-GG-3X sites, it was found that despite a much higher oxidation potential Ž; 2.0 V vs. NHE. repair of the T -) T dimer proceeded much more efficiently than did oxidation of the 5X-GG-3X site Ž1.0 V vs. NHE for 5X-G.. This was ascribed to the much faster rate of repair for the T -) T dimer in relation to reaction rates associated with generation of the base–labile lesion at 5X-GG-3X . The kinetic preference for T -) T dimer repair appeared to override the thermodynamic preference for 5X-GG-3X damage resulting from oxidation potential differences. We also examined the effect on long range oxidation of binding a M. HhaI mutant methylase containing a substitution of glutamine to tryptophan mutation. In wild-type Žwt. M. HhaI, the glutamine at position 237 is inserted within the base stack cavity created by base flipping. Thus, conversion of this glutamine to the flat aromatic heterocycle tryptophan was anticipated to exert an interesting influence upon long range charge transfer through the protein-bound DNA. In this case, with the aromatic side chain of the tryptophan inserted in the helix, long range oxidation was restored. Hence DNA binding proteins can not only inhibit but can also facilitate long range charge transport. Interestingly, DNA interaction with this tryptophan mutant of M. HhaI gave rise also to damage at the central 5X-UG-3X step Žconsistent with DNA-enzyme binding. as well as very intense alkali–labile damage at the 5X-G of the binding site 5X-GUGC-3X ; this damage was negligible upon binding of wt. M. HhaI. Rudimentary molecular modeling of the energy minimized tryptophan mutant bound to the native binding sequence 5X-GCGC-3X suggested that insertion of tryptophan within the p-gap

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may afford extensive p-stacking with the 5X-G at which damage is observed. That protein–DNA interactions in this manifold may mimic those molecular interactions responsible for 5X-G selectivity in oxidative damage at 5X-GG-3X steps suggests possibilities of far-reaching consequence. Particularly intriguing is that modulation of nucleobase redox potential by protein binding events could represent a means by which DNA damage processes are attenuated. Alternatively, might redox potential differences within DNA, either free or associated with specific binding proteins, be exploited by enzymes as a means of molecular recognition andror signaling?

5. A role for DNA-mediated redox chemistry in damage recognition: some speculations We have therefore seen in chemical model systems how damage might be sensed locally using recognition based upon helix destabilization, and even at a distance, using DNA-mediated charge transport chemistry. Recognition which exploits local helix destabilization as an element of binding using either thermodynamic or kinetic features can clearly successfully mimic some of the features of a DNA repair enzyme. But can they mimic all of the features? Specifically, are substrate recognition and the levels of efficiency estimated to be needed to search the genome satisfied by mechanisms based upon helix destabilizing recognition alone? Could some sensing of DNA damage be accomplished at least in part based upon DNA-mediated charge transport chemistry? It is possible that nature may have evolved DNA sequences which passively serve to protect regions of the genome from long range radical migration. We have seen for example that 5X-TA-3X steps may be considered as somewhat insulating. Perhaps also G-rich stretches might represent hot spots to which radical damage is directed. But might DNA electron transfer also be exploited more actively through the binding and reactions of proteins bound to the helix. We have, for example, already seen that proteins which bind to DNA can modulate chemical transformations at a distant site based upon DNA charge transport. DNA binding

proteins not only can inhibit but also can activate DNA electron transfer chemistry. Are these strategies exploited in nature? Many examples of DNA repair proteins which contain electroactive cofactors exist. Perhaps the prototypical example is DNA photolyase w130–133x. In effecting repair of T -) T dimers, the enzyme exploits the two electron-reduced cofactor FADHy as the electron source, although modeling studies have shown that repair of T -) T dimers may actually proceed in some cases via electron transfer from an active site tryptophan directly to the dimer w134x. There currently are no data to suggest that T -) T dimers are repaired in a reaction involving long range charge migration. In contrast to DNA photolyase, the BER proteins Endonuclease IIIŽEndoIII. and MutY contain w4Fe–4Sx2q clusters located proximally to the proposed points of DNA–enzyme contact w68–70,135x. Fe–S clusters are ubiquitous in and often serve as electron sinks for biological redox processes w136x. As summarized in Table 1, MutY and EndoIII display radically different substrate specificities. Interestingly, the w4Fe–4Sx 2q clusters within both enzymes are proposed to play structural roles since efforts to oxidize or reduce the cluster in either protein have failed. Nonetheless, it has been shown that denatured MutY can very efficiently refold into the ‘‘near-native’’ stable conformation in the absence of the w4Fe–4Sx 2q cluster, suggesting little, if any, contribution to overall structural stability w137x. Yet what is remarkable is that the cluster of MutY is absolutely critical for DNA recognition. Thus any assignment of a strictly structural role for the MutY w4Fe–4Sx 2q cluster may be premature. In this context, the increasing prevalence of DNA repair proteins which contain Fe–S clusters is intriguing. In addition to playing key roles in DNA repair, redox chemistry is also of critical importance in transcriptional regulation. Transcription is frequently coupled to redox states of bound proteins, as with SoxR w138x and FNR w139–141x proteins. Redoxsensitive DNA binding has also been observed for the JunrFos, CREBrATF, p53, NF-Y, AP-1 and Nf-kB transcription factors and in many cases is coupled to thiol-disulfide interchange w142x. Additionally, it has been shown that transcription is often attenuated by redox active DNA repair proteins such as apurinic endonuclease ŽAPErRef-1. w143,144x.

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Indeed this subtle interplay of the functioning of redox active enzymes suggests that DNA fullfils more than just the classically perceived role of a library. What follows are proposals for how DNA sensing using charge transport might be exploited by naturally occurring enzymes. These are based upon our current understanding of DNA-mediated electron transfer, as well as the workings of redox-sensitive macromolecular interactions. No data in support of these mechanisms have yet been obtained. These ideas may be viewed instead as starting points for inquiry around which meaningful experiments may be designed. We limit ourselves to two basic mechanisms. The first relies upon a processive scanning motif coupled with long range charge transport. Importantly, the scheme takes advantage of DNA-mediated charge transport in providing a novel means to scan large regions of the genome. Hence, for each single point of DNA–protein contact, many more base pairs may be read than would be possible using surface contacts alone. The second view illustrates another attribute of DNA electron transfer chemistry. Here we exploit the electronic characteristics of DNA lesions and their differentiation from the native DNA nucleobases by differences in redox reactivity.

65

This dissociation of the oxidized enzyme would then allow its relocation to another region of DNA. Reduction and reactivation of processive scanning capabilities of this enzyme could then occur, either through diffusional contacts with the new region of DNA Žand subsequent reduction from a distance., or enroute to the new DNA substrate, presumably through direct enzyme–enzyme or enzyme-reductant contacts. This cycle of procession followed by oxidation, translocationrreduction, and re-oxidation would afford a highly efficient way to scan the genome. In effect, redox chemistry through the p-stack allows a much larger number of bases to be scanned than would be possible with a strictly processive mode of sequence reading. In the absence of DNA disruptions, long range oxidation proceeds quite readily. However, as envisioned in panel B ŽScheme 1., disruptions in base stacking which do not allow oxidation state changes would restrict the enzyme to procession down the helix to the site of the local disruption. Without DNA-mediated electron transfer which facilitates dissociation and the sampling of new sites, the enzyme is forced to close in on the aberrant site. Arrival at the site of base pair disruptionrdamage could then lead to direct enzymatic action or to the assembly of a multi-subunit repair complex as illustrated.

5.1. ProcessiÕity leading to charge transfer signaling 5.2. Electron transfer with a redox-sensitiÕe lesion Many repair enzymes probably scan for sites of damage at least in part in a processive fashion. However, strictly processive manifolds of scanning would appear to be too slow and inefficient in keeping the 10 9 base pairs of the human genome intact, particularly in light of the rate at which nucleic acid damage occurs. Might the processive action of repair enzymes therefore be supplemented by DNA electron transfer scanning? In such a manifold ŽScheme 1, panel A. one could envision procession of a given protein down the DNA double helix, with concomitant long-range redox interactions with another redox active protein at some distance mediated by the p-stack. Electron transfer affords the net oxidation of one, and reduction of the other. As is common with numerous transcription factors, oxidation of this enzyme could then effect its dissociation from the DNA substrate.

Significant differences in redox potential of DNA base lesions from those of the native bases are common. A particularly noteworthy example is that of the prevalent oxidative lesion 8-oxo-2X-deoxyguanosine Ž E8 ; 0.7 V vs. NHE. versus deoxyguanosine Ž E8 s 1.29 V vs. NHE. w145x. Even within native DNA, significant sequence-dependent variations in oxidation potentials of the DNA bases have been noted; the E8 for the 5X-G of a 5-GGG-3X site is about 0.5 V lower than for a lone G w119–121x. Moreover such differences in redox potential have been shown to bear profound influence upon longrange DNA damage processes w146x. Thus enzymes bound to DNA could react either from a distance or locally with such lesions by way of redox processes. Moreover, as illustrated earlier in the competition between oxidation of T -) T dimers and 5X-GG-3X

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Scheme 1. Processive scanning mechanism coupled with long-range electron-transfer based sensing of disruptions in p-stacking of DNA base pairs.

doublets, these reactions could be under kinetic rather than thermodynamic control. Such reactions can promote changes in the enzyme or site, making recognition and repair of the DNA lesion more facile.

Thus, as shown in Scheme 2, one might envision that for each DNA-enzyme encounter, the enzyme injects either an electron or an electron deficient hole Ždepending upon the redox potentials available.

S.R. Rajski et al.r Mutation Research 447 (2000) 49–72

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Scheme 2. Long-range electron transfer as a means of detecting basesrlesions with modified redox potential relative to the native nucleotides.

within the p-stack. Although normally returned to its native oxidation state upon binding to undamaged DNA Žeither by cross-talk with another enzyme, or by back electronrhole transfer., the incorporation of an electron or hole sink within the base pair stack could give rise to permanent oxidation state changes in the protein. Additionally, the electron hole could react within the base stack. A change in protein oxidation state may then signal a conformational transition, giving rise to local processive scanning of the DNA for damage or perhaps the recruitment of other DNA repair associated factors ŽRAFs. which lack scanning activity but are highly efficient at DNA repair. Clearly, the redox potential of modified DNA sites could also play a large role in damage recognition, enhancing differences in the lesion transiently from unmodified DNA.

6. Conclusions Efforts by many research groups have yielded considerable insight into the issues governing how repair enzymes might recognize and react at sites of

DNA damage. From these efforts, it has become quite clear that the structures of mismatches and lesions lead often to significant changes in the thermodynamic and kinetic stabilities of damaged base pairs. Such structural and dynamic trademarks may help enzymes in targeting damage sites, and indeed enzyme action itself may serve to enhance and promote DNA conformational changes, including the pinching, pulling and flipping out of bases from the DNA polymer. But are such processes sufficient to account for the level of screening for DNA damage that must function within the cell? One such screening strategy could take advantage of DNA-mediated electron transfer chemistry. This seems particularly appealing in view of the high degree of efficiency with which such charge transfer reactions occur. We have found that DNA-mediated charge transport can occur over long molecular distances and is exquisitely sensitive to DNA base stacking and structure. Hence this chemistry provides an approach to detect perturbations or aberrations in DNA, and indeed to do so from a distance. Moreover, redox-triggered alterations could yield powerful macromolecular signals;

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certainly redox-triggered processes are critical in transcriptional regulation. Can we envision a role for DNA charge transport in facilitating these biological processes? As we move into a new millenium of discovery, there is little doubt that these mechanisms for damage recognition should be elucidated. Hopefully, the strategies and approaches described here will stimulate new experiments and perspectives, so that we might begin to unravel the remarkable chemistry which underlies how repair proteins recognize damage in DNA.

Acknowledgements We are grateful to the NIH for their financial support of our research. In addition we thank the American Cancer Society Žto S.R.R.., the National Science Foundation Žto B.A.J.., and the Parsons Foundation Žto B.A.J.. for fellowship support. We are also grateful to Dr. R.J. Melamede for providing preprints detailing work in his laboratories at University of Vermont.

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