Molecular Dynamic Simulations of Cisplatin- and Oxaliplatin-d(GG) Intrastand Cross-links Reveal Differences in their Conformational Dynamics

doi:10.1016/j.jmb.2007.07.079 J. Mol. Biol. (2007) 373, 1123–1140

Molecular Dynamic Simulations of Cisplatin- andOxaliplatin-d(GG) Intrastand Cross-links RevealDifferences in their Conformational Dynamics

Shantanu Sharma1, Peng Gong1, Brenda Temple2

Debadeep Bhattacharyya1, Nikolay V. Dokholyan1,2

and Stephen G. Chaney1,2⁎

1Department of Biochemistryand Biophysics, School ofMedicine, University ofNorth Carolina, Chapel Hill,NC 27599-7260, USA2Lineberger ComprehensiveCancer Center, University ofNorth Carolina, Chapel Hill,NC 27599, USA

Received 15 May 2007;received in revised form26 July 2007;accepted 28 July 2007Available online23 August 2007

*Corresponding author. E-mail addrAbbreviations used: CP, cisplatin;

cyclobutanedicarboxylatoplatinum(Imolecular dynamics; HMG, high mobeta; hpol η, human DNA polymera

0022-2836/$ - see front matter © 2007 E

Mismatch repair proteins, DNA damage-recognition proteins and transle-sion DNA polymerases discriminate between Pt-GG adducts containing cis-diammine ligands (formed by cisplatin (CP) and carboplatin) and trans-RR-diaminocyclohexane ligands (formed by oxaliplatin (OX)) and thisdiscrimination is thought to be important in determining differences inthe efficacy, toxicity and mutagenicity of these platinum anticancer agents.We have postulated that these proteins recognize differences in conforma-tion and/or conformational dynamics of the DNA containing the adducts.We have previously determined the NMR solution structure of OX-DNA,CP-DNA and undamaged duplex DNA in the 5′-d(CCTCAGGCCTCC)-3′sequence context and have shown the existence of several conformationaldifferences in the vicinity of the Pt-GG adduct. Here we have usedmolecular dynamics simulations to explore differences in the conforma-tional dynamics between OX-DNA, CP-DNA and undamaged DNA in thesame sequence context. Twenty-five 10 ns unrestrained fully solvatedmolecular dynamics simulations were performed starting from twodifferent DNA conformations using AMBER v8.0. All 25 simulationsreached equilibrium within 4 ns, were independent of the starting structureand were in close agreement with previous crystal and NMR structures.Our data show that the cis-diammine (CP) ligand preferentially formshydrogen bonds on the 5′ side of the Pt-GG adduct, while the trans-RR-diaminocyclohexane (OX) ligand preferentially forms hydrogen bonds onthe 3′ side of the adduct. In addition, our data show that these differences inhydrogen bond formation are strongly correlated with differences inconformational dynamics, specifically the fraction of time spent in differentDNA conformations in the vicinity of the adduct, for CP- and OX-DNAadducts. We postulate that differential recognition of CP- and OX-GGadducts by mismatch repair proteins, DNA damage-recognition pro-teins and DNA polymerases may be due, in part, to differences in thefraction of time that the adducts spend in a conformation favorable forprotein binding.

© 2007 Elsevier Ltd. All rights reserved.

Keywords: cisplatin; oxaliplatin; molecular dynamics; simulations;conformation
Edited by D. Case
ess: [email protected](II); CBDCA, carboplatin; cis-diammine-1,1-I); OX, oxaliplatin; trans-R,R-diaminocyclohexaneoxalatoplatinum(II); MD,bility group; RMSD, root-mean-square deviation; hpol β, human DNA polymerasese eta; PCA, principal component analysis.

lsevier Ltd. All rights reserved.

mailto:[email protected]

1124 MD Simulations of CP- and OX-DNA Adducts

Introduction

Cisplatin (CP; cis-diamminedichloroplatinum(II))and carboplatin (CBDCA,cis-diammine-1,1-cyclobu-tanedicarboxylatoplatinum(II)) are widely used fortreatment of testicular cancer, ovarian cancer, headand neck tumors and a variety of other solid tumors.However, many tumors are intrinsically resistant ordevelop acquired resistance to these chemothera-peutic agents, and tumors that are resistant to one ofthese two platinum compounds are usually cross-resistant to the other. The mutagenicity of CP invivo1 is also of concern because secondary malignan-cies have been associated with CP chemotherapy.2

Considerable effort has been made to develop thirdgeneration platinum anticancer agents that wouldnot share these limitations. Oxaliplatin (OX; trans-R,R-1,2-diaminocyclohexaneoxalatoplatinum(II)) isone such compound, and has recently been ap-proved for the treatment of colorectal cancer andtumors that are resistant to CP and CBDCA. WhileOX does have some mutagenicity,3 it appears to beless mutagenic than CP.4 CP and CBDCA formPt-DNA adducts that contain the cis-diamminecarrier ligands, while OX forms Pt-DNA adductsthat contain the trans-RR-1,2-diaminocyclohexanecarrier ligand. For simplicity, we will refer to theseas CP-DNA and OX-DNA adducts throughout thispaper. Other than the differences in carrier ligand,the adducts formed by CP, CBDCA and OX appearto be identical in terms of the type of adduct formed(60–65% intrastrand GG, 25–30% intrastrand AG,5–10% intrastrand GNG and 1–3% interstrand) andthe site of adduct formation.5–7

Because of their abundance, the intrastrand GGadducts are thought to be major determinants of thecytotoxic response to platinum anticancer agents.The basis for the differences in tumor range andmutagenicity of OX compared to CP and CBDCA isnot known, but is thought to be determined by theability of proteins involved in damage recognition,damage repair and/or damage tolerance to discri-minate between CP and OX adducts. For example,hMSH2 and MutS bind with greater affinity to CP-GG adducts than to OX-GG adducts8,9 and, as mightbe expected from this difference in binding affinity,defects in mismatch repair result in resistance toCP and CBDCA, but not to OX.8,10–13 Similarly, anumber of damage recognition proteins and tran-scription factors, especially those with high mobilitygroup (HMG) domains, have been shown to dis-criminate between CP- and OX-GG adducts.14,15

The mechanism(s) by which the binding of theseproteins to Pt-DNA adducts influences the cytotoxicresponse is not known, but has been postulatedto involve shielding of the adducts from DNA re-pair and tolerance mechanisms,16–19 activation ofsignaling pathways leading to cell cycle arrest orapoptosis, and/or hijacking of transcription factorsneeded for DNA replication or cell division.15,20 Thebinding specificity has been determined for only afew of these proteins, but where it has been studiedthese proteins bind to CP-GG adducts with higher

affinity than to OX-GG adducts.14,15,21 Finally,translesion DNA polymerases such as hpol β andhpol η have been shown to bypass OX-GG adductswith higher efficiency than CP-GG adducts,19,22,23

which might contribute to the differences in CP andOX mutagenicity.The CP- and OX-GG adducts form in the major

groove and bend the DNA in the direction of themajor groove. The proteins that discriminatebetween CP- and OX-GG adducts either bind tobent DNA or bend the DNA in the direction of themajor groove after binding.24–27 Because these pro-teins primarily interact with the minor groove, wehave hypothesized that the ability of the proteinsto discriminate between CP- and OX-GG adductsprobably results from subtle differences in con-formation or conformational dynamics in the DNAcontaining the two adducts rather than fromphysical interaction of the proteins with the carrierligands of the adducts in the major groove. Anumber of structures have been reported for CP-GGand OX-GG adducts. The overall conformation ofDNA containing these adducts appears to besimilar, but exact comparisons have been difficultto make because the structures have been deter-mined by different techniques (crystallographyversus NMR), in different sequence contexts andwith oligonucleotides of different length. The NMRstructures obtained to date have varied with respectto the number and resolution of NMR constraintsobtained and the molecular mechanics simulationsused to convert the NMR constraints to final struc-tures.28–30 X-ray crystallographic structures havebeen reported for the CP-GG and OX-GG adducts inthe same sequence context,31,32 but these structuresmay have been constrained by crystal packingrestraints. We have recently obtained high resolu-tion NMR solution structures of the OX-GG,30 CP-GG adducts33 and undamaged DNA33 in the samemutagenic AGGC sequence context (the underlinedbases indicate the position of the Pt-GG adduct). TheNMR studies have identified several conformationaldifferences between the solution structures of thetwo Pt-GG adducts. Here we have used moleculardynamics (MD) simulations to extend this analysisto differences in conformational dynamics betweenCP-GG adducts, OX-GG adducts and undamagedDNA in the AGGC sequence context. Our data areconsistent with earlier reports of greater distortionon the 5′ side of Pt-GG adducts.29,30,34–39 Our dataare also consistent with previous reports that the cis-diammine (CP) ligand preferentially forms hydro-gen bonds on the 5′ side of the Pt-GG adduct, whilethe trans-RR-diaminocyclohexane (OX) ligand pre-ferentially forms hydrogen bonds on the 3′ side ofthe adduct. Finally, our data show that these dif-ferences in hydrogen bond formation are stronglycorrelated with differences in conformational dy-namics, specifically the fraction of time spent indifferent DNA conformations, for CP- and OX-DNAadducts. These differences were particularly evidentfor propeller twist, buckle, slide and shift in thevicinity of the Pt-GG adduct. We postulate that these

Figure 1. Average RMSD values for the MD simula-tions over time. The RMSD values for each of the 10simulations compared to the corresponding NMR solutionstructure30,33 for the CP-DNA (a) and OX-DNA adducts(b) are shown for the full 10 ns of each simulation. RMSDat time t represents the average of RMSD from time zero totime t. CP-NMR and OX-NMR represent simulationsstarting from NMR solution structures of the Pt-DNA12-mer duplexes. CP-SRY and OX-SRY stand for simula-tions starting from the more distorted DNA–SRY struc-tures. The five simulation trajectories starting from CP-NMR and OX-NMR structures with different initial MDvelocities are represented as continuous lines (circlesymbol), dotted lines (square symbol), broken lines(diamond symbol), long broken lines (upward trianglesymbol) and dot-dashed lines (left triangle symbol). Thefive simulation trajectories starting from the more dis-torted CP-SRYandOX-SRY structures with different initialMD velocities are represented as continuous lines (down-ward triangle symbol), dotted lines (right triangle sym-bol), broken lines (plus symbol), long broken lines (crosssymbol) and dot-dashed lines (asterisk symbol).

1125MD Simulations of CP- and OX-DNA Adducts

differences in conformational dynamics could allowdifferential recognition of CP- and OX-GG adductsby critical DNA-binding proteins that influence thecytotoxic response to these adducts. For example,we postulate that CP-GG adducts spend a greaterpercentage of time in conformations favorable forbinding of mismatch repair and HMG-domainDNA-binding proteins, while OX adducts spend agreater percentage of time in conformations(s) favo-able for bypass by hpol β and hpol η. Experimentsare currently underway to test these hypotheses.Experiments are also underway to compare theeffect of sequence context on the conformationaldifferences between these two adducts.

Results

The MD simulations were independent ofstarting structure

In total, twenty-five 10 ns unrestrained fullysolvated molecular dynamics simulations wereperformed starting from five different DNAconformations. In order to critically evaluatewhether the starting structures influenced theMD simulations, two very different startingstructures were used for both the CP-DNA andOX-DNA simulations. One of the starting struc-tures for the CP- and OX-DNA simulations usedthe NMR structure of DNA complexed with hSRY(Protein Data Bank accession code 1J46, bendangle=54°) for the DNA backbone. The otherstarting structures were based on the NMRstructures of the CP-DNA (Protein Data Bankaccession code 2NMW; bend angle=22°) and OX-DNA (Protein Data Bank accession code 1PGC;bend angle=31°) adducts alone. Idealized B-DNAwas used as the starting structure for the B-DNAsimulation. Five simulations each were performedwith the CP-SRY, CP-NMR, OX-SRY, OX-NMR andB-DNA starting structures as described in Methods.The all-atom mass-weighted root-mean-squaredeviations (RMSDs) referenced to the correspondingNMR structures for the CP-DNA and OX-DNAstructures and the starting B-DNA structure forundamaged DNA calculated over all 25 trajectorieswere used to determine how long it took thesimulations to reach equilibrium, and, in turn, therange of trajectories to be used for the subsequentanalyses. Plots of RMSD over time are shown forCP-DNA (Figure 1(a)), OX-DNA (Figure 1(b)) and B-DNA (Supplementary Data, Figure S1). The averageRMSD plots of all ten CP-DNA simulations werewithin close proximity of one another within thefinal 6 ns. Thus, the simulations of the CP-DNAadducts converged to similar equilibrium structureseven when the starting structures differed signifi-cantly in the initial degree of DNA bending and/orMD initial velocity. Similar behavior was evident forthe OX-DNA adducts. Based on these plots ofaverage RMSD over time, we concluded that thesimulations reached equilibrium within 4 ns. There-

fore our comparisons were made based on the final6 ns of the trajectories. The average RMSD comparedto theNMR solution structures30,33 was 2.08(±0.43) Åover all ten simulations for OX-DNA, 2.73(±0.53) Åover all ten simulations for CP-DNA and 2.80(±0.30)Å over all five simulations for B-DNA.

Comparison of the MD simulations withpreviously reported structures

For the purposes of comparison with previousstructures, centroid structures (the structure fromthe simulation with the lowest RMSD compared to


the average structure) were determined for both theCP-DNA and OX-DNA simulations. Figure 2 showsan overlay of the CP-DNA and OX-DNA centroidstructures and “sausage diagrams” of each structureindividually showing the overall variation in thestructures during the simulations. The centroidstructures of the CP-DNA and OX-DNA adductswere very similar (the all-atom RMSD for the DNAportion of the structures was 1.3 Å). The sausagediagrams showed that the greatest variations in boththe CP-DNA and OX-DNA structures occurred atthe ends of the DNA molecules, but there wasrelatively little fluctuation of the DNA backboneduring the simulations.

Figure 2. Average CP-DNA and OX-DNA structures weroverall average structure was computed over the equilibratedsimulations starting with the CP-SRY, CP-NMR and OX-SRY,performed for the SRY and NMR starting structures. Becauselittle influence on the average structure (RMSD=0.83 Å forOX-NMR), the two average structures were further averaged tosimulations of the CP-DNA and OX-DNA adducts. KabschOX-DNA average structures against every 1 ps snapshot ofsimulation snapshots having lowest RMSD with corresponstructures. (a) Overlay of CP-DNA and OX-DNA centroid(c) “Sausage diagrams” of CP-DNA and OX-DNA centroidtuations of the CP-DNA and OX-DNA phosphate backbone wthe 30,000 snapshots of equilibrated final 6 ns of all ten simulattool was used to compute these fluctuations. The backbonecorresponds to the relative fluctuations of CP-DNA (b) and O

The centroid structures of the CP-DNA and OX-DNA simulations were also compared with theprevious crystal31,32 and NMR30,33 structures of thesame adducts (Figure 3). In both cases, the simula-tions agreed slightly better with the NMR structuresthan the crystal structures. For example, the all atomRMSD comparisons of the CP-DNA centroid struc-ture with the corresponding crystal31 and NMR33

structures were 4.2 Å and 3.1 Å, respectively.Similarly, the all-atom RMSD comparisons of theOX-DNA centroid structure with the correspondingcrystal32 and NMR30 structures were 4.0 Å and3.1 Å, respectively. For the central four base-pairs,the RMSD values were 3.5 Å, 2.8 Å, 3.6 Å and 2.9 Å

e obtained using the AMBER ptraj tool. In each case, thefinal 6 ns trajectories combined for the five independentOX-NMR, respectively. Pair-wise Kabsch alignment wasthe RMSD values indicated that the starting structure hadCP-SRY versus CP-NMR and 1.03 Å for OX-SRY versusobtain a single average structure for the last 6 ns of all tenalignment was then performed against CP-DNA andsimulation trajectory. The CP-DNA and OX-DNA MDding average structures were selected as the centroidstructures. Cyan, OX-DNA; purple, CP-DNA. (b) andstructures, respectively. Root-mean-square atomic fluc-ere obtained by averaging the backbone fluctuations overion trajectories. The atomicfluct routine in the AMBER ptrajthickness and color (in a scale varying from blue to red)X-DNA (c) backbone phosphate groups.

Figure 3. Comparison of CP-DNA and OX-DNA centroid structures with previously determined crystal and NMRstructures. Pair-wise Kabsch alignment of the centroid structure (cyan) obtained from the CP-DNA and OX-DNAsimulations versus the corresponding crystal structures (red) and NMR structures (orange). The NMR solution structuresof CP-DNA33 and OX-DNA30 (Protein Data Bank entries 2NPW and 1PG9, respectively) in the d(CCTCAGGCCTCC)-3′sequence context were used to compare against corresponding centroid structures. The crystal structures correspondingto CP-DNA31 and OX-DNA32 were derived from the available structures in the 5′-d(CCTCTGGTCTCC)-3′ sequencecontext (Protein Data Bank entries 1AIO and 1IHH, respectively). Representative crystal structures in the 5′-d(CCTCAGGCCTCC)-3′ sequence context were derived bymutating the nucleotide sequence using Tripos Sybyl. Pair-wiseKabsch alignment of the CP-DNA and OX-DNA centroid structures was performed using PyMOL's pair_fit tool.


for the CP-crystal versus CP-centroid, CP-NMRversus CP-centroid, OX-crystal versus OX-centroidand OX-NMR versus OX-centroid, respectively.In order to further assess how well the MD

simulation structures agreed with our previouslyreported NMR structures,30,33 the extent to which

the MD simulations reproduced NMR-derivedinter-proton distances was also evaluated. Therewere 171 and 160 inter-proton distance constraintsderived from NMR data within the central fourbase-pairs 5′-d(A5G6G7C8)-3′ of CP-DNA and OX-DNA adducts, respectively.30,33 The statistics of the


inter-proton distance violations (defined as an inter-proton distance deviating from the range of corre-sponding distance constraints by more than 0.5 Å;see Methods) are listed in Table 1 and the details ofthese distance violations are listed in SupplementaryData, Table S1. Overall, there were two (b2%) andfive (b3%) violations for OX-DNA and CP-DNA,respectively. Most of the violations in CP-DNAsimulation structures and one of the two violationsin OX-DNA simulation structures involved A5 andG6 nucleotides, which were on the 5′ side of theadduct and may represent the intrinsic dynamicsof this portion of the molecule. Furthermore, one ofthe OX-DNA violations and three of the CP-DNAviolations involved sugar protons, and may repre-sent differences in sugar pucker, rather than dif-ferences in the position of the purine or pyrimidinebases. The very small number of violations com-pared to the total number of distance constraintsindicated that our MD simulations largely repro-duced the NMR data.The geometry of the Pt-DNA adducts was also

compared with previously reported structures forthe CP- and OX-DNA adducts (SupplementaryData, Table S2). The platinum displacement fromthe plane of the central guanine bases and theplatinum out of guanine plane bending angles weremuch closer to the previously reported values forthe CP- and OX-DNA adducts than was the recentMD simulation of a CP-DNA adduct reported byElizondro-Riojas & Kozelka,40 possibly reflectingthe refinement of partial charges that we derivedand utilized for these simulations. Our MD simula-tions do not reflect the differences between CP- andOX-DNA adducts with respect to platinum displa-cement from the plane of the central guanine basesand the platinum out of guanine plane bendingangles that were seen in our previous NMR solutionstructures of the adducts30,33 (Supplementary Data,Table S2). However, it is important to note that theposition of the platinum is not directly determinedby NMR constraints in the solution structures andthat the CNS software with the partial chargesreported earlier by Yao et al.41 and Scheef et al.42 wasused to derive the lowest energy solution structures.Thus, this discrepancy between our MD simulations

Table 1. Violations of NMR constraints for the central four ba

Base-base protons

CP-DNA (171) Interstrand 1Intrastrand 1

Same nucleotide 0OX-DNA (160) Interstrand 0

Intrastrand 1Same nucleotide 0

Inter-proton differences averaged from the final 6 ns of the simueffect (NOE) derived inter-proton differences for CP-DNA andOdistances from the MD simulations trajectories, which differed from thconsidered violations. The total number of inter-proton distance constthe first column. Restraints are reported according to category in thebetween base and base protons or between base and sugar pucker pr

and the solution structures in the geometry of theplatinum adduct may simply reflect differences instructure refinement, such as partial charges ordifferences between the AMBER and the CNSsoftwares used for calculations of the NMR struc-tures. AMBER and CNS use different force fieldsand the AMBER simulation was performed underunrestrained and fully solvated condition, whereasCNS calculation used the simulated annealing pro-tocol in vacuum and was restrained by the inter-proton distance constraints.

Principal component analysis of majorconformational dynamics

Principal component analysis can be used tosegregate large-scale correlated motions from ran-dom thermal fluctuations, thereby probing theessential dynamics of the system. Principal compo-nent analysis (PCA) is an orthogonal lineartransformation that maps the data to a newcoordinate system so that the system can bedeconvoluted along these coordinates. The orderof coordinates is rank-ordered according to theircontribution to the motion of the system. Principlecomponent analysis was used to analyze thetrajectories from each set of simulations for majorconformational motions. The first three principalcomponents described N90% of the essential modesof dynamics for the platinum-adducted DNAcomplexes. As expected for DNA simulations, thefirst three components of conformational motionsroughly corresponded to a superposition of bend-ing (Supplementary Data, movie S1), twisting(Supplementary Data, movie S2) and winding(Supplementary Data, movie S3) motions, in thatorder. By superimposing the major motions for CP-and OX-DNA adducts (Supplementary Data,movies 1–3) it is evident that the overall con-formational flexibility of DNA containing CP-GGand OX-GG adducts is very similar.

Hydrogen bonds

To assess the stability of the DNA duplex, theoccupancy of all possible hydrogen bonds (calculated

se-pairs

ViolationsTotal

constraintsBase-pucker protons Total violations

0 1 181 2 432 2 1100 0 181 2 440 0 98

lation trajectories were compared to the nuclear OverhauserX-DNA30,33 as described inMethods. All average inter-protone NOE inter-proton distance constraints by more than 0.5 Å, wereraints for the central four base-pairs is reported in parentheses inlast column. The middle columns give the number of violationsotons, and the total number of violations in each category.

Figure 4. Hydrogen bond occupancy of the centralfour base-pairs. The hydrogen bond occupancy of thecentral four base-pairs was calculated as described inMethods. The standard Watson–Crick hydrogen bondoccupancy of base-pairs for B-DNA (circle), CP-DNA(square) and OX-DNA (triangle) are shown.


as the percentage of time during the simulation thatthe hydrogen bonds existed), was measured for CP-DNA and OX-DNA adducts and B-DNA of the samesequence. The data for Watson–Crick hydrogen bondoccupancy are shown in Figure 4. When compared toB-DNA, both CP-DNAandOX-DNAadducts show asignificant decrease in standardWatson–Crick hydro-gen bond occupancy for the A5•T20 and G6•C19

Figure 5. Hydrogen bonds between platinum carrier ligabond formation between Pt-amine hydrogen atoms and the sadduct (left) and the OX-DNA adduct (right). The DNA backPt-DNA adduct and darker gray for the complementary stranmaroon. For the CP adduct, all amine hydrogen atoms are in eamine hydrogen atoms are not in equilibrium. The equatorial hin light blue. The broken lines illustrate the potential hydrogenbases.

base-pairs on the 5′ side of the adduct, whereas thebase pairs on the 3′ side of the adduct are almostcompletely intact.Two hydrogen bonds with significantly high

occupancy between the platinum carrier ligandand the DNA were identified and are shown inFigure 5. One is between the 3′ amine hydrogen ofthe platinum and the oxygen atom O6 of the 3′ G7and the other is between the 5′ amine hydrogen ofthe platinum and the nitrogen atom N7 of A5. Forthe CP-DNA adduct the amine hydrogen atoms areequivalent so only four combinations of hydrogenbond formation are possible. The occupancy of eachof these hydrogen bond combinations is summar-ized in Table 2A. Differences in global DNAconformation associated with 5′ only, 5′ and 3′,and 3′ only hydrogen bonds were minimal. How-ever, significant conformational differences wereobserved in the central four base-pair region andthese are shown in Supplementary Data, Figure S2.The CP-DNA adduct spends 40.2% of its time inconformations that allow formation of the 5′hydrogen bond only and 13.3% of its time inconformations that allow formation of the 3′hydrogen bond only. However, the CP-DNA adductalso spends a significant amount of time (34.0%) inconformations that allow formation of both the 5′and 3′ hydrogen bonds. The total occupancy of the5′ and 3′ hydrogen bonds for the CP-DNA adduct is74.2% and 47.2%, respectively. Thus, for the CP-DNA adduct the occupancy is greater for the hy-drogen bond on the 5′ side of the adduct, suggesting

nds and DNA. Structures illustrating observed hydrogenurrounding DNA bases are shown for both the CP-DNAbone is shown in light gray for the strand containing thed. The Pt adduct including the carrier ligand is shown inquilibrium and are shown in cyan. For the OX adduct theydrogen is shown in cyan and the axial hydrogen is shownbonds between the platinum amine groups and the DNA


that the CP-DNA adduct is preferentially orientedtowards the 5′ side.For the OX-DNA adduct the situation is some-

what more complicated because the axial andequatorial amine hydrogen atoms are not equiva-lent (Figure 5). Thus, on the 5′ side of the adduct,the hydrogen bond between the axial hydrogenand N7 of A5 is not equivalent to the hydrogenbond between the equatorial hydrogen and N7 ofA5. However, on the 3′ side of the adduct only theequatorial hydrogen is in sufficient proximity tothe O6 of G7 to form a hydrogen bond. Thus, forthe OX-DNA adducts there are six possiblecombinations of hydrogen bond formation. Theoccupancy of each is summarized in Table 2B. Asseen for the CP-DNA adduct, differences in globalDNA conformations associated with these hydro-gen patterns were minimal. The differences inDNA conformation in the central four base-pairregion associated with 5′ equatorial only, both 5′equatorial and 3′ equatorial and 3′ equatorial onlyhydrogen bonds are shown in SupplementaryData, Figure S3. The OX-DNA adduct spends34.2% of its time in conformations that allowformation of the 3′ hydrogen bond only and 13.7%of its time in conformations that allow formation of5′ hydrogen bonds only. However, it also spends asignificant amount of time (40.9%) in conforma-tions that allow simultaneous formation of both ahydrogen bond between the equatorial hydrogenon the 5′ side and N7 of A5 and a hydrogen bondbetween the equatorial hydrogen on the 3′ side andO6 of G7. The total occupancy of the 5′ and 3′hydrogen bonds for the OX-DNA adduct is 58.1%and 78.7%, respectively. Thus, for the OX-DNAadduct the occupancy is greater for the hydrogenbond on the 3′ side of the adduct, suggesting thatthe OX-DNA adduct is preferentially orientedtowards the 3′ side.

Table 2. Occupancy of hydrogen bonds between platinum am

Pattern of H bond formation

5′ Side

A. CP-DNA adductPt-amine-H • A5 Pt-

++––

B. OX-DNA adductPt-amine-axial-H • A5 Pt-amine

++

Pt-amine-equitorial-H • A5 Pt-amine++

No Pt-amine-H • A5 Pt-amine––

The percentage occupancy of hydrogen bonds between Pt-amine hydras described in Methods.

DNA conformational dynamics

While the overall centroid structures of the CP-DNA and OX-DNA simulations were very similar,we observed significant differences between the twosimulations in terms of DNA conformational dy-namics. To determine the effect of CP- and OX-GGadducts on the conformational dynamics of DNA,the frequency distributions (fraction of the timespent in each conformation) from the trajectoriesof the CP-DNA, OX-DNA and undamaged DNAsimulations were calculated using the programCURVES v5.3. From previous comparisons ofCP- and OX-DNA adducts, it appeared that theywere most likely to differ in terms of overall bendangle and the DNA helical parameters of the centralfour base pair region.30–33 The overall bend anglewas calculated from the CURVES output usingMadBend (http://monod.biomath.nyu.edu). No sig-nificant differences in the frequency distribution ofbend angles was observed for the CP- and OX-DNAadduct simulations (data not shown). The frequencydistributions of DNA helical parameters for thecentral four base pairs were taken directly from theCURVES output and were analyzed for statisticalsignificance by the Kolmogorov–Smirnov test43

(Supplementary Data, Table S3). The Kologorov–Smirnov test determines how significantly twodistributions differ from each other, withoutmakingany assumption regarding the distribution of data(non-parametric and distribution-free). Cases inwhich the frequency distributions of DNA helicalparameters were significantly different from unda-maged DNA for both CP- and OX-DNA adducts areindicated in bold in Supplementary Data, Table S3.The distribution of frequency values for those helicalparameters between both types of Pt-DNA adductsand undamaged B-DNA are shown in Figure 6.When comparing both Pt-DNA adducts to B-DNA,

ine hydrogen atoms and nearby bases on the DNA

Occupancy ofH bond (%)3′ Side

amine-H • G7– 40.2+ 34.0+ 13.3– 12.5

-equitorial-H • G7– 7.7+ 3.5

-equitorial-H • G7– 6.0+ 40.9

-equitorial-H • G7+ 34.3– 7.6

ogen atoms and near-by DNA bases (see Figure 5) was calculated

mailto:[email protected]

Figure 6. Frequency distributions of representative DNA duplex helical parameters for the central four base-pairs:differences between Pt-DNA adducts and undamaged DNA. Frequency distributions of each DNA duplex helicalparameter for the central four base-pairs were calculated from the simulation trajectories by CURVES v5.3 as described inMethods. The frequency distribution histograms were calculated from the structures obtained at every picosecond overthe final 6 ns of each equilibrated MD simulation. Thus, the histograms for CP-DNA and OX-DNA adducts were derivedfrom 60,000 structures, while the histograms for undamaged DNA were derived from 30,000 structures. Selectedfrequency distributions that show differences between Pt-DNA adducts and undamaged DNA are shown. B-DNA(dotted lines with diamond symbols); CP-DNA (continuous lines with circle symbols); OX-DNA (long broken lines withsquare symbols). (The symbols do not represent the data ponts; rather they are shown to help distinguish the curves.)


there were several striking differences identified,including buckle and propeller twist for the A5·T20base-pair, shear and propeller twist for the G6·C19base-pair, buckle for the G7·C18 base-pair; roll at theG6-G7 base-pair step, and slide, tilt and roll at theG7-C8 base-pair step. These differences indicatedthat the conformational dynamics profile of B-DNAwas altered by the platinum adducts and areconsistent with the previously reported conforma-tional distortions imposed on B-DNA by Pt-GGadducts.28,29,31,32,44 At the G6-G7 base-pair step, theprofiles of frequency distribution of roll were almostidentical for CP-DNA and OX-DNA adducts, whichwas consistent with the GGdihedral angles for thesetwo adducts.The significance of differences in the frequency

distributions of DNA helical parameters between

CP- and OX-DNA adducts was also analyzed by theKolmogorov–Smirnov test43 (Supplementary Data,Table S4). The distributions of those helical para-meters showing the greatest difference between CP-and OX-DNA adducts are shown in Figure 7, alongwith the distribution pattern of undamaged B-DNAfor comparison. When comparing CP-DNA adductsto OX-DNA adducts, there were some noticeabledifferences. For base-base helical parameters, differ-ences were observed for propeller twist for theG6·C19 and G7·C18 base-pairs and buckle for theC8·G17 base-pair. For base-pair step helical para-meters, differences were observed for slide at theA5-G6, G6-G7 and G7-C8 base-pair steps and shift atthe G7-C8 base-pair step. All of the above differ-ences suggest that CP-DNA and OX-DNA adductsdiffer in conformational dynamics.

Figure 7. Frequency distributions of representative DNA duplex helical parameters for the central four base-pairs:differences between CP-DNA and OX-DNA adducts. Frequency distributions of each DNA duplex helical parameter forthe central four base-pairs were calculated from the simulation trajectories by CURVES v5.3 as described in Methods. Thefrequency distribution histograms were calculated from the structures obtained at every picosecond over the final 6 ns ofeach equilibrated MD simulation. Thus, the histograms for CP-DNA and OX-DNA adducts were derived from 60,000structures, while the histograms for undamaged DNA were derived from 30,000 structures. Selected frequencydistributions that show differences between CP-DNA and OX-DNA adducts are shown. B-DNA (dotted lines withdiamond symbols), CP-DNA (continuous lines with circle symbols); OX-DNA (long broken lines with square symbols).(The symbols do not represent the data points; rather they are shown to help distinguish the curves.) The vertical dash-dotlines show the value of the corresponding DNA helical parameter on the 3′ side of the adduct in the crystal structure of theHMG–CP-DNA adduct.26


Correlation between platinum amine hydrogenbond formation and DNA conformationaldynamics

As described above the percentage occupancy forhydrogen bonds between the Pt-amine hydrogensand nearby bases was greater on the 5′ side for theCP-DNA adduct and the 3′ side for the OX-DNAadduct. The data described above indicated that theCP- and OX-DNA adducts primarily influenced theconformational dynamics of the central four base-pairs. Thus, to determine whether the conforma-tional dynamics of CP- and OX-DNA adducts mightbe influenced by the formation of these hydrogenbonds, the trajectory data for DNA helical para-meters for the central four base pairs were separatedaccording to the patterns of hydrogen bond forma-

tion (Figures 8 and 9). The resulting frequencydistributions were analyzed for significance by theKolmogorov–Smirnov test43 (Supplementary Data,Tables S5 and S6) and cases in which the frequencydistribution of DNA helical parameters associatedwith 5′ only hydrogen bonds were significantlydifferent than the DNA helical parameters asso-ciated with 3′ only hydrogen bonds are indicatedin bold in Supplementary Data, Tables S5 and S6.The large number of examples in which the fre-quency distribution of DNA helical parameters isassociated with the pattern of hydrogen bondformation (5′ only versus 3′ only) suggests that theconformational dynamics of many of the DNAhelical parameters in the central four base-pairregion are strongly correlated with the pattern ofhydrogen bond formation.

Figure 8. Effect of hydrogen bonding patterns on frequency distributions of selected DNA duplex helical parametersfor the central four base-pairs of CP-DNA adducts. The effect of hydrogen bond patterns between the Pt-amine hydrogenatoms and adjacent DNA bases on the frequency distributions of each DNA duplex helical parameter for the central fourbase-pairs of the CP-DNA adduct were determined as described in Methods. The frequency distribution histograms wereobtained from all of the CP-DNA structures containing a particular hydrogen bond pattern (Table 2). Thus, the histogramsfor 5′ only, 5′ plus 3′ and 3′ only hydrogen bonds were obtained from 24,120, 20,400 and 7980 structures, respectively.Selected frequency distributions that show difference in the distribution of DNA helical parameters when the hydrogenbond exists on the 5′ side compared to when the hydrogen bond exists on the 3′ side of the CP-DNA adduct are shown. 3′Hydrogen bond only (continuous lines with square symbols); 5′ hydrogen bond only (dotted lines with diamondsymbols); hydrogen bonds on both 3′ and 5′ sides (long broken lines with upward triangle symbols). (The symbols do notrepresent the data points; rather they are shown to help distinguish the curves.) The frequency distributions for nohydrogen bonds on either the 3′ side or 5′ side are not shown for ease of viewing.


Selected examples for the CP-DNA adduct areshown in Figure 8. For most of the DNA helicalparameters shown there is a clear difference in thedistribution of DNA helical parameters when thehydrogen bond exists on the 5′ side compared towhen the hydrogen bond exists on the 3′ side. In thecase of the CP-DNA adduct, the two major hydro-gen bond patterns are hydrogen bond on 5′ sideonly (40.2%) and hydrogen bonds on both the 5′ and3′ side (34.0%). Furthermore, the frequency distribu-tions of DNA helical parameters associated withthese two hydrogen bond patterns are generallyeither almost identical (A5-G6 slide) or very similar(G6•C19 propeller twist, G7•C18 propeller twist,C8•G17 buckle, G6-G7 slide) G7-C8 shift, G7-C8slide). These data suggest that DNA conformationsin which the hydrogen bonds are seen on both the 5′and 3′ side may represent conformational transi-

tions between the 5′ only and 3′ only hydrogen bondconformations.For the OX-DNA adduct (Figure 9 and Supple-

mentary Data, Table S6) there also appear to be cleardifferences in the distribution of DNA helicalparameters when the hydrogen bond exists on the5′ side only (either for the axial hydrogen or theequatorial hydrogen) compared to when the hydro-gen bond exists on the 3′ side only. In the case of theOX-DNA adduct, the two major hydrogen bondpatterns are a hydrogen bond with the equatorialhydrogen on 3′ side only (34.3%) and hydrogenbonds with both the equatorial hydrogen on the 5′and the equatorial hydrogen on the 3′ side (40.9%).As with the CP-DNA adduct, the frequency dis-tributions of DNA helical parameters associatedwith these two hydrogen bond patterns are gen-erally either almost identical (G6-G7 twist, G7-C8

Figure 9. Effect of hydrogen bonding patterns on frequency distributions of selected DNA duplex helical parametersfor the central four base-pairs of OX-DNA adducts. The effect of hydrogen bond patterns between the Pt-amine hydrogenatoms and on adjacent DNA bases on the frequency distributions of each DNA duplex helical parameter for the centralfour base-pairs of the OX-DNA adduct were determined as described in Methods. The frequency distribution histogramswere obtained from all of the OX-DNA structures containing a particular hydrogen bond pattern (Table 2). Thus, thehistograms for 5′ axial only, 5′ equatorial only, 5′ axial plus 3′ equatorial, and 3′ equatorial only hydrogen bonds wereobtained from 3600, 4620, 24540 and 20500 structures, respectively. Selected frequency distributions that show differencein the distribution of DNA helical parameters when the hydrogen bond exists on the 5′ side compared to when thehydrogen bond exists on the 3′ side of the OX-DNA adduct are shown. 3′ Equatorial hydrogen bond only (continuouslines with square symbols); 5′ axial hydrogen bond only (dot-dashed lines with circle symbols); 5′ equatorial hydrogenbond only (dotted lines with diamond symbols); hydrogen bonds with both 3′ equatorial and 5′ equatorial hydrogens(long broken lines with upward triangle symbols). (The symbols do not represent the data points; rather they are shown tohelp distinguish the curves.) The frequency distributions for no hydrogen bonds on either the 3′ side or 5′ side andhydrogen bond formation for both the 3′ equatorial and 5′ axial hydrogen atoms are not shown for ease of viewing.


shift, G7-C8 slide) or very similar (G6•C19 propellertwist, G7•C18 propeller twist, C8•G17 buckle, G6-G7 slide). These data again suggest that DNAconformations in which the hydrogen bonds areseen on both the 5′ and 3′ side may representconformational transitions between the 5′ only and3′ only hydrogen bond conformations.Finally, when one compares the data in Figures 8

and 9 with the data in Figure 7 it becomes apparentthat most of the conformational differences asso-ciated with hydrogen bond formation in the CP-and OX-DNA adducts are highly correlated withthe conformational differences seen between CP-and OX-DNA adducts in Figure 7. For example,when one looks at propeller twist at the G6•C19base-pair, the two major distribution patterns

associated with 5′ hydrogen bond formation (5′only and both 5′ and 3′) of the CP-DNA adduct(representing 74.2% of the total hydrogen bondoccupancy) are centered at −18° to −16° (Figure 8),while the two major distribution patterns asso-ciated with 3′ hydrogen bond formation (3′ onlyand equatorial 5′ plus 3′) for the OX-DNA adduct(representing 75.2% of the total hydrogen bondoccupancy) are centered at −29° and −21° (Figure9). This precisely accounts for the difference infrequency distribution patterns seen between CP-and OX-DNA adducts (−18° for CP and −25° forOX) for G6•C19 propeller twist in Figure 7. Similarcomparisons can be made for G7•C18 propellertwist, C8•G17 buckle, A5-G6 slide, G6-G7 slide,and G7-C8 slide.


However, these patterns are not universal. For G7-C8 shift, the differences in distribution patternsbetween CP- and OX-DNA adducts in Figure 7correlate with the major distribution patterns for CP-and OX-DNA adducts in Figures 8 and 9, but thedifferences between 5′ only and 3′ only hydrogenbond conformational distributions are not significantfor either CP- or OX-DNA adducts (SupplementaryData, Tables S5 and S6). At the opposite extreme, the5′ only and 3′ only hydrogen bond conformationaldistributions are significantly different for G6-G7twist (Supplementary Data, Tables S5 and S6)), butthe major distribution patterns (5′ only and both 5′and 3′ for CP and 3′ only and both 5′ axial and 3′ forOX) are very similar (22–26° for CP and 28° for OX).Thus, the distribution patterns for CP- and OX-DNAadducts for G6-G7 twist are not significantlydifferent (Supplementary Data, Table S4).

Discussion

Accuracy of the MD simulations

Partial atomic charges for the platinum atom andthe surrounding atoms of Pt-GG adducts are notavailable in the standard AMBER force field andmust, therefore be developed ab initio. The pre-viously available charges for CP-GG adducts wereadopted by Yao et al.41 from the ab initio calculationsof the [Pt(NH3)3(Ade)]2+ complex by Kozelka et al.45

over 13 years ago. The same atomic charge pa-rameters were used by Scheef et al.42 for simulationsof the OX-GG adduct, with the sole exception thatthe charge on the amine nitrogen was modified toreflect its attachment to the cyclohexane ring.Although the partial charge parameters on the CP-GG adduct have been updated by Elizondo-Riojas &Kozelka,40 those charge parameters were not actu-ally reported. Because the partial charge parametersfor the CP-GG adduct were not derived empiricallyfor a Pt-GG adduct and the OX-GG partial chargeparameters had never been determined empirically,we have developed new partial charge parametersfor both CP-GG and OX-GG adducts. The newpartial charges on the platinum atomwere similar tothe previous published values.40,41 However, thenew partial charges on the surrounding nitrogenatoms were significantly different. Therefore, it wasimportant to validate the accuracy of our newpartial charges in MD simulations.Our MD simulations converged to a common set

of structures within the first 4 ns that were inde-pendent of the starting structure and MD initialvelocities and these structures reproduced thecrystal and NMR solution structures of the sameadducts in DNA by several criteria. First, the simu-lation structures in the final 6 ns had an averageRMSD of b3 Å with respect to our NMR solutionstructures (Figure 1). Second, the centroid structureshad RMSD values of around 4.1 Å compared to thecorresponding crystal structures31,32 and around3.1 Å compared to the corresponding NMR struc-

tures.30,33 Third, for the central four base-pair regionthere were less than 3% violations of NMR-derivedinter-proton distance constraints for the CP-GG andOX-GG adducts and the majority of these violationsinvolved sugar pucker protons. Finally, both CP-DNA and OX-DNA adducts in our MD simulationsshowed significantly increased roll angle at theG6•G7 base–pair step and the GG dihedral anglewith respect to the undamaged B-DNA, which isconsistent with both the crystal and NMR solutionstructures of the Pt-DNA adducts.29–33 By all ofthese criteria, the MD simulations were excellentrepresentations of both the crystal and NMRsolution structures of the same adducts. In addition,these MD simulations provided a better estimationof the α angles and displacements of the platinumatom out of the 5′ and 3′ guanine planes than theprevious MD simulation of the CP-GG adduct byElizondo-Riojas & Kozelka.40

The DNA duplex is more distorted on the 5′ sideof the adduct than on the 3′ side

We found a significantly greater decrease ofoccupancy of standard Watson–Crick hydrogenbonds for the 5′ A5•T20 and 5′ G6·C19 base-pairsthan for the 3′G7·C18 base-pair for both CP-GG andOX-GG adducts, which suggests that the Pt-GGadduct is more distorted on the 5′ side of the adductthan on the 3′ side of the adduct. This is consistentwith a number of biological and structural studiesthat have indicated that Pt-GG adducts are moredistorted on the 5′ side of the adduct than on the 3′side of the adduct. For example, several authorshave reported that the majority of misinsertionmutations occur opposite the 5′G of CP-GGadducts,34–36 and we and others have shown thathpol η has significantly greater difficulty extendingthe DNA chain past the 5′G than the 3′G of eitherCP-GG or OX-GG adducts.37–39 With respect tostructural studies, Marzilli et al.29 have reported afaster exchange rate between the water and iminoprotons of the 5′G of CP-GG adducts and we haveshown a faster exchange rate between water andimino protons of the 5′G of both the OX-GG30 andCP-GG adducts.33 Marzilli et al.29 also reported anunusually large positive shift and slide at 5′ X/Gbase pair-step, and Elizondo-Riojas & Kozelka,40 intheir MD simulations, have shown greater mobilityof 5′G·C base-pair with respect to the base-pair 5′ tothe Pt-GG adduct. All of these previous studies haveindicated that the 5′G·C base-pair was more flexibleand/or more distorted than the 3′ G·C base-pair.

Orientation of CP-DNA and OX-DNA adducts

In the crystal structure of CP-DNA adduct, ahydrogen bond was reported between the hydrogenatom of the 5′ oriented ammine NH3 ligand and anoxygen atom of the phosphate group on the 5′ sideof the Pt-GG adduct.31 In contrast, in the crystalstructure of OX-DNA adduct, a hydrogen bond wasfound between the 3′ oriented amine NH2 group of


the diaminocycloheaxane ligand and the oxygenatom O6 of the 3′G.32 Thus it has been suggestedthat the cis-diammine carrier ligand of cisplatinwas oriented more towards the 5′ side of the adductand the (trans-R,R)1,2-diaminocyclohexane carrierligand of oxaliplatin was oriented more towardsthe 3′ side of the adduct. However, neither hydro-gen bond was observed in NMR solution structuresof the CP- and OX-GG adducts.28,30,33 Here, weassessed the occupancy of these hydrogen bondsfor both CP-DNA and OX-DNA adducts and foundthat the occupancy of the hydrogen bond betweenthe 5′-oriented NH3 and the phosphate oxygenreported in the crystal structure of the CP-DNAadduct was less than 1% for both CP-DNA and OX-DNA adducts (Supplementary Data, Figure S4). Intheir work based onMD simulations of the CP-DNAadduct, Elizondo-Riojas & Kozelka40 also reportedthat the formation of this hydrogen bond is a veryrare event, unless one considers the possibility ofhydrogen bonds bridged by one or two water mole-cules. The fact that this hydrogen bond is not ob-served at any significant level in either NMRsolution structures or MD simulations suggeststhat the formation of such a hydrogen bond is arare event and that the crystal structure of the CP-DNA adduct happened to capture this rare con-formation, possibly due to crystal packing.However, we did observe significant occupancy of

the hydrogen bond formed between the hydrogenatom of the 5′ ammine NH3 ligand of CP or the 5′amine NH2 group of diaminocyclohexane ligand ofOX and the nitrogen atom N7 of A5. The occupancyof this hydrogen bond was higher for the CP-DNAadduct (74.2%) than the OX-DNA adduct (58.1%).We also observed significant occupancy of the hy-drogen bond formed between the hydrogen atom ofthe 3′ ammine NH3 ligand of CP or the 3′ amineNH2 group of diaminocyclohexane ligand of OXand the oxygen atom O6 of the 3′ G7, which hadbeen previously reported in the crystal structure ofthe OX-DNA adduct, but not the CP-DNA adduct.The occupancy of this hydrogen bond was higherfor the OX-DNA adduct (78.7%) than the CP-DNAadduct (47.2%). The differences in occupancy ofhydrogen bonds between the ammine/aminehydrogen atoms of the CP/OX and the bases onthe 5′ and 3′ side of the Pt-GG adducts suggest adifference in the orientation of Pt carrier ligandswith respect to the DNA between CP- and OX-GGadducts. The cis-diammine carrier ligand of CPappears to be oriented more towards the 5′ side ofthe adduct and the (trans-R,R)1,2-diaminocyclohex-ane carrier ligand of OX appears to be oriented moretowards the 3′ side of the adduct. Thus, our data areconsistent with the observations reported by Lip-pard and colleagues31,32 with respect to the prefer-ential orientation of carrier ligands of CP-DNA andOX-DNA adducts. This preferential formation ofhydrogen bonds on the 5′ side of the CP-DNAadducts and on the 3′ side of OX-DNA adducts ishighly correlated with differences in conformationand conformational dynamics of DNA containing

CP- and OX-GG adducts. We postulate that thehydrogen bond formation between the Pt-aminehydrogen atoms and adjacent bases drives some ofthe important conformational differences betweenCP- and OX-DNA adducts.These experiments do not address the possible

effect of sequence context on the orientation ofCP- and OX-GG adducts. Our MD simulationswere done in the AGGC sequence context, whilethe crystal structures reported by Lippard and col-leagues31,32 were in the TGGT sequence context,which suggests that the relative orientation ofCP- and OX-GG adducts are similar in those se-quence contexts. However, in the MD simulations ofthe CP-GG adduct in the CGGA sequence context byElizondo-Riojas & Kozelka,40 a hydrogen bond wasobserved on the 3′ side of the adduct and betweenthe 3′ NH3 ligand of the CP-GG adduct and N7 ofthe A on the 3′ side of the adduct. This observationmay help explain previous reports that an A on the3′ side of Pt-GG adducts has a significant affect onthe binding affinity and binding specificity of DNAdamage recognition proteins for CP-GG adducts.Experiments are currently underway to determinethe effect of sequence context on the orientation andconformational dynamics of CP- and OX-GGadducts.

Differences in conformational dynamicsbetween CP-DNA and OX-DNA adducts

From the comparison of frequency distributionof DNA duplex helical parameters, we observedseveral differences which appear to reflect the dis-tinct conformational dynamics between CP-DNAandOX-DNA adducts.While the total range of DNAconformations explored by CP- and OX-GG adductsis very similar, the fraction of time spent in theseconformation by CP- and OX-GG adducts differedwith respect to several DNA helical parameters inthe vicinity of the Pt-GG adducts. We postulate thatthese differences in conformational dynamics couldallow differential recognition of CP- and OX-GGadducts by critical DNA-binding proteins that in-fluence the cytotoxic response to these adducts.While the differences in conformational dynamicsbetween CP- and OX-DNA adducts are relativelysmall, they are fully consistent with the 1.5- totwofold differences in recognition of the adducts bymost of the DNA-binding proteins studied to date.We postulate that CP-GG adducts spend a greater

percentage of time in conformations favorable forbinding of mismatch repair and HMG-domainDNA-binding proteins, while OX adducts spend agreater percentage of time in conformations(s)favorable for bypass by hpol β and hpol η. Forexample, the HMG domain of both HMG-A1 andHMG-B1 has been shown to bind with higher ef-ficiency to CP-GG adducts than to OX-GG adducts14and the crystal structure of the HMG–CP-DNAcomplex has been reported.26 Since HMG binds tothe CP-DNA adduct on the 3'side of the adductonly,26 the DNA helical parameters on the 3′ side of


the HMG–CP-DNA complex are shown as verticalbroken lines in Figure 7 for comparison with thefrequency distributions of helical parameters for theCP-GG and OX-GG adducts. For propeller twist ofthe G7•C18 base-pair, buckle of the C8•G17 base-pair and shift and slide at the G7-C8 base-pair step,the CP-GG adduct appears to spend a greater per-centage of its time in a conformation that is similarto the conformation of the HMG–CP-GG complexthan the OX-GG adduct. If any of these conforma-tions are characteristic of the conformation of theprotein–DNA complex in the initial recognitionstep, they might facilitate the recognition of theCP-GG adduct by the HMG domain. The confor-mational dynamics differences at the G7-C8 base-pair step might be particularly critical because boththe affinity of the HMG domain for the CP-GGadduct14,46,47 and the ability of the HMG domain todiscriminate between CP-GG and OX-GG adducts14

is highly dependent on the base to the 3′ side of theadduct.There are a number of important limitations

inherent in such comparisons. We are comparing acrystal structure of the HMG–CP-GG DNA complexwith unrestrained simulations of the CP-GG andOX-GG DNA adducts; the final, stable protein DNAcomplex likely has a different conformation from theinitial protein–DNA recognition complex; and thesequence context of the CP-GG adduct in the HMG–CP-GG complex was different from the one in theCP-GG and OX-GG simulations. To better charac-terize those conformational differences that areimportant for differential protein recognition,experiments are underway to simulate the CP-GGand OX-GG adducts in the same AGGC sequencecontext in complex with the HMG domains of oneor more proteins that discriminate between theadducts.

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Methods

Starting structures

All five starting structures contained a 12-mer DNAduplex in the same sequence context of 5′-d(CCTCAGGCCTCC)-3′ for the strand containing theplatinum adduct. For the DNA portion of the structures,the NMR solution structures of the OX-DNA adduct30

(Protein Data Bank accession code 1PGC) and the CP-DNA adduct33 (Protein Data Bank accession code 2NPW)solved in our laboratory were used as two of the startingstructures. For the CP- and OX-DNA adducts, the DNAportion of the NMR solution structure of DNA complexedwith hSRY (Protein Data Bank accession code 1J46) wasalso used as a starting structure for the DNA backbone.The original DNA sequence from 1J46 was mutated to thesame DNA sequence used for our NMR solutionstructures. Since the DNA backbone of the hSRY–DNAcomplex was more bent than the DNA in the NMRsolution structures of the Pt-DNA adducts by themselves,it provided a test of whether our MD simulations werecapable of driving significantly distinct starting struc-tures to non-distinguishable structures when simulations

reached equilibrium. For the platinum adducts on theDNA backbone derived from the hSRY–DNA complex,the oxaliplatin adduct was obtained from our NMRsolution structure of OX-GG30 (1PGC) and the cisplatinadduct was obtained from the NMR solution structure ofthe CP-GG adduct reported by Marzilli et al.29 (1KSB).The Pt-GG adducts were superimposed on the DNAfrom the hSRY–DNA complex, with removal of the twoG bases of the adduct producing the starting structures ofthe CP- and OX-DNA adducts. For undamaged DNA,ideal B-DNA was used as the starting structure. Themolecular modeling package InsightII† was used toperform all manual structural preparation and building,including the undamaged DNA and the DNA sequencechange for the CP- and OX-DNA adducts.

Force field parameterization

The atomic partial charges for CP-GG and OX-GGadducts are not defined in the standard AMBER forcefield library. However, they are required and crucialin MD simulations. In order to determine the atomicpartial charges of CP-GG and OX-GG adducts, the 9-methyl-guanine derivatives cis- [Pt(NH3)2(9-Me-Guo)2]

2+

(CP-meG2) and [Pt(trans-RR-1,2-diaminocyclohexane)(9-Me-Guo)2]

2+ (OX-meG2) were used to simplify thecalculation. These derivatives were manually built fromour NMR solution structures using Insight II. The atomicpartial charges were determined using the Mullikenmethod implemented within Gaussian03 based on eitherthe structure geometry optimized by Gaussian03 or theNMR structure modified to the 9-methyl-guanine deri-vative. The density functional method B3LYP implemen-ted within Gaussian03 was utilized; the LanL2DZ basisset was used for the platinum atom and 6-31Gd basis setwas used for the rest of the atoms. The Mulliken methodwas found to be insensitive to the geometry of thestructure and the resulting atomic partial charges wereconsistent in both geometry optimized and non-opti-mized structures.The partial charge of the platinum atomwas close to the

previously published values, while the partial charges offour nitrogen atoms surrounding the platinum atom weresignificantly different from the published values. Thepartial charges of the rest of the atoms were within thetheoretical range and comparable with their counterpartsin the standard AMBER force field. The Mulliken chargesbased on the structure of the original 9-methyl-guaninederivative modified from our NMR solution structurewere used without further geometry optimization as thenew atomic partial charges for Pt-GG adducts (Supple-mentary Data, Table S7) and incorporated into our forcefield parameters. The atomic partial charges of chemicallyequivalent atoms (such as the pseudo-equatorial hydro-gen atoms of the ammine group and the correspondingatoms in the two guanine bases) were averaged and thesmall charge discrepancy due to the structural differencebetween 9-methyl-guanine and deoxyguanine was dis-tributed to the sugar according to the standard chargetransfer technique used by Yao et al.41 Besides the atomicpartial charges, other force field parameters of the Pt-GGadducts were referenced from AMBER parm99 force fieldparameters or from previous work by Yao et al.41 andScheeff et al.42 The MD simulation protocol is described inSupplementary Data.

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MD simulations and trajectory analysis

Altogether 25 10 ns unrestrained and fully solvated MDsimulations were carried out by using SANDERmodule ofAMBER v8.0. There were five replicas for each of the fivestarting structures described above. The five replicasdiffered only in the MD initial velocity assigned whenthe system was heated for the last time. The atomiccoordinates of the structures were saved every 1 ps. Boththe 5′ and 3′-terminal base-pairs were excluded fromanalysis because in several simulations the terminal baseswere not base-paired and in some extreme cases they werestacked with each other. In contrast with previous MDsimulations of Pt-DNA adducts, we did not use artificialconstraints to hold the terminal base-pairs together.Therefore only the central ten base-pairs were consideredin trajectory analysis. The PTRAJ module of AMBER v8.0was employed for trajectory analysis.

Principal component analysis

The ptraj program from the AMBER v8 simulationsoftware was used to perform PCA. The final 6 ns ofequilibrated MD simulations of each of the 20 trajectoriesof CP-DNA and OX-DNA were subject to PCA analysis.The first three principal components described N90% ofthe essential modes of dynamics for the platinum-adducted DNA complexes. The dynamics of the firstthree principal components were visualized using thePyMOL molecular graphics tool‡.

Hydrogen bond occupancy

A distance of less than 3.5 Å and an angle of greater than120° between the potential hydrogen bond donor andacceptor were used as the criteria for a hydrogen bondformation. The occupancy of one hydrogen bond wasdefined as the percentage of frames in which the hydrogenbond existed. The hydrogen bond occupancy of one base-pair was defined as the average occupancy of hydrogenbonds existing within this base-pair. For example, thehydrogen bond occupancy of normal G·C base-pair wasthe average occupancy of the three standard Watson–Crick hydrogen bonds. However, there were hydrogenbonds formed between non-complementary bases indamaged DNA adducts because of the distortion andmisalignment of bases. When calculating the hydrogenbond occupancy of base-pairs, the occupancy of such non-complementary hydrogen bonds of at least 5% wasnecessary for consideration and the occupancy was splitevenly between two base-pairs.

Inter-proton distance constraint comparison

A set of inter-proton distance constraints have beenderived from NMR data and used to compute the NMRsolution structures for the OX-DNA and CP-DNA30,33 byCNS (crystallography and NMR system) program. In theCNS calculations, a distance violation is defined as aninter-proton distance deviating from the range ofcorresponding distance constraints by more than 0.5 Å.The same criterion was applied here when we tried toevaluate how well our MD simulation structures agreedwith the NMR data. In addition, we classified these

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constraints into several categories based on the locationsof two protons. The two protons may be from the samenucleoside, from two nucleosides within the same strand(intrastrand) or from two nucleosides on differentstrands (interstrand). For each proton, it may be a baseproton or a sugar pucker proton. The H1′ proton wastreated as a base proton because its position is largelyindependent of sugar pucker. All the other protons ofthe sugar were considered as sugar pucker protonsbecause their positions were strongly influenced by thesugar pucker conformation. Only the distance constrainsof the central four base pairs 5′-d(A5G6G7C8)-3′ wereconsidered in this study.

DNA helical parameter analysis of trajectories

Five independent simulations with randomized initialvelocities were performed for each of the followingstarting conformations: CP-SRY (the CP-GG adduct,1KSB, superimposed on the DNA structure of thehSRY–DNA complex, 1J46), OX-SRY (the OX-GG adduct,1PGC, superimposed on the DNA structure of thehSRY–DNA complex, 1J46), CP-NMR (the NMR struc-ture of the CP-DNA 2NPW), OX-NMR (the NMRstructure of the OX-DNA adduct, 1PGC) and B-DNA(constructed with Insight II§). The ptraj tool fromAMBER-8∥ was used to extract the equilibrated con-formations between 4 ns and 10 ns of simulation time,recording snapshots at every 1 ps time-interval of thefive independent runs of AMBER simulation trajectories.Using ptraj these trajectory snapshots were saved in theProtein Data Bank (PDB) format, resulting in a total of(10,000–4000)×5 snapshots, i.e. 30,000 snapshots for eachof CP-SRY, OX-SRY, CP-NMR, OX-NMR and B-DNAsimulations.Each nucleotide type was converted from AMBER

format to PDB format, and the resulting snapshots weresubjected to CURVES analysis. The following CURVESparameters were extracted: global inter base-pair para-meters: shift, slide, rise, tilt, roll and twist; global base-base parameters: shear, stretch, stagger, buckle, propellerand opening. Histograms were then constructed forpercent occupancy versus discrete units of each DNAhelical parameter. Initially, for both CP-DNA and OX-DNA adducts histograms were constructed for theCURVES parameters obtained with each starting struc-ture (NMR and SRY) individually to determine whetherthe starting structure affected the distribution of CURVESparameters. Except for slight differences observed forshift at the A5-C6 base-pair step, the CURVES parameterswere not influenced by starting structure (data notshown). For CP-DNA and OX-DNA simulations, theCURVES trajectories for both the SRY and NMR startingstructures were combined to get better statistics offluctuations in the CURVES parameters (60,000 snapshotseach). Percentage occupancy distributions of the DNAhelical parameters were calculated by normalizing thefrequency distributions to 100%.The Kolmogorov–Smirnov test43 was performed for

each helical parameter to calculate the P value fordifferences between CP-DNA versus OX-DNA adducts(Supplementary Data, Table S5) and between B-DNA andboth CP-DNA and OX-DNA adducts (Supplementary

§www.acelrys.com∥Case et al. (2004). University of California, San

Fransisco.

http://www.pymol.org

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Data, Table S4). The Kologorov–Smirnov test determineshow significantly two distributions differ from each other,without making any assumption regarding the distribu-tion of data (non-parametric and distribution-free). Forease of comparison P values were reported on a negativelogarithmic scale, -log(P) such that larger values implygreater statistical difference (Supplementary Data, TablesS4 and S5). In order to determine which of these -log(P)values were significant, the Kolomogorov–Smirnov testwas also performed for the same helical parameterscomparing five individual simulations of the samestructure (i.e. five for CP-DNA, five for OX-DNA andfive for B-DNA). The highest -log(P) value obtained fromcomparisons of the same structure was considered to setthe threshold of significance. Only −log(P) values for thecomparisons between CP-DNA and OX-DNA or betweenB-DNA and both CP-DNA andOX-DNA that were greaterthan the −log(P) value for comparisons of the samestructures were considered significant.

Correlation of patterns of hydrogen bondformation with DNA helical parameters

A trajectory-wide binary profile of all combinations ofthe presence and absence of the following hydrogen bondswas generated using the hydrogen bond occupancyprocedure described above: CP-DNA: 5′ side: 5′ Pt-amine hydrogen to 5N7; 3′ side: 3′ Pt-amine hydrogen to7O6; and OX-DNA: 5′ side: 5′ Pt-amine equatorialhydrogen to 5N7; 5′ Pt-amine axial hydrogen to 5N7; 3′side: 3′ Pt-amine equatorial hydrogen to 7O6. DNA helicalparameters of the corresponding trajectory snapshotswere recorded using the helical parameter analysisprocedure described above. Histograms were then con-structed for percent occupancy versus discrete units ofeach DNA helical parameter for each of these hydrogen-bond combinations. The Kolmogorov–Smirnov test43 wasperformed for each helical parameter to calculate P valuesfor the differences between 5′ hydrogen bond only and 3′hydrogen bond only for both the CP-DNA (Supplemen-tary Data, Table S6) and the OX-DNA adducts (Supple-mentary Data, Table S7). The significance of these P valueswas determined as described above.

Acknowledgement

This work was supported by USPHS grantCA84480.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2007.07.079

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Molecular Dynamic Simulations of Cisplatin- and Oxaliplatin-d(GG) Intrastand Cross-links Reveal Differences in their Conformational Dynamics