Efficient and error-free replication past a minor-groove N2-guanine adduct by the sequential action of yeast Rev1 and DNA polymerase zeta

Rev1, a member of the Y family of DNA polymerases, functions in lesion bypass together with DNA polymerase zeta (Pol zeta). Rev1 is a highly specialized enzyme in that it incorporates only a C opposite template G. While Rev1 plays an indispensable structural role in Pol zeta-dependent lesion bypass, the role of its DNA synthetic activity in lesion bypass has remained unclear. Since interactions of DNA polymerases with the DNA minor groove contribute to the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, here we examine the possibility that unlike other DNA polymerases, Rev1 does not come into close contact with the minor groove of the incipient base pair, and that enables it to incorporate a C opposite the N(2)-adducted guanines in DNA. To test this idea, we examined whether Rev1 could incorporate a C opposite the gamma-hydroxy-1,N(2)-propano-2'deoxyguanosine DNA minor-groove adduct, which is formed from the reaction of acrolein with the N(2) of guanine. Acrolein, an alpha,beta-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from other oxidation reactions. We show here that Rev1 efficiently incorporates a C opposite this adduct from which Pol zeta subsequently extends, thereby completing the lesion bypass reaction. Based upon these observations, we suggest that an important role of the Rev1 DNA synthetic activity in lesion bypass is to incorporate a C opposite the various N(2)-guanine DNA minor-groove adducts that form in DNA.     

Replication past a trans-4-hydroxynonenal minor-groove adduct by the sequential action of human DNA polymerases iota and kappa

The X-ray crystal structure of human DNA polymerase iota (Poliota) has shown that it differs from all known Pols in its dependence upon Hoogsteen base pairing for synthesizing DNA. Hoogsteen base pairing provides an elegant mechanism for synthesizing DNA opposite minor-groove adducts that present a severe block to synthesis by replicative DNA polymerases. Germane to this problem, a variety of DNA adducts form at the N2 minor-groove position of guanine. Previously, we have shown that proficient and error-free replication through the gamma-HOPdG (gamma-hydroxy-1,N2-propano-2'-deoxyguanosine) adduct, which is formed from the reaction of acrolein with the N2 of guanine, is mediated by the sequential action of human Poliota and Polkappa, in which Poliota incorporates the nucleotide opposite the lesion site and Polkappa carries out the subsequent extension reaction. To test the general applicability of these observations to other adducts formed at the N2 position of guanine, here we examine the proficiency of human Poliota and Polkappa to synthesize past stereoisomers of trans-4-hydroxy-2-nonenal-deoxyguanosine (HNE-dG). Even though HNE- and acrolein-modified dGs share common structural features, due to their increased size and other structural differences, HNE adducts are potentially more blocking for replication than gamma-HOPdG. We show here that the sequential action of Poliota and Polkappa promotes efficient and error-free synthesis through the HNE-dG adducts, in which Poliota incorporates the nucleotide opposite the lesion site and Polkappa performs the extension reaction.     

Human DNA polymerase iota promotes replication through a ring-closed minor-groove adduct that adopts a syn conformation in DNA

Acrolein, an alpha,beta-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from oxidation of polyamines. The reaction of acrolein with the N2 group of guanine in DNA leads to the formation of a cyclic adduct, gamma-hydroxy-1,N2-propano-2'-deoxyguanosine (gamma-HOPdG). Previously, we have shown that proficient replication through the gamma-HOPdG adduct can be mediated by the sequential action of human DNA polymerases (Pols) iota and kappa, in which Poliota incorporates either pyrimidine opposite gamma-HOPdG, but Polkappa extends only from the cytosine. Since gamma-HOPdG can adopt either a ring-closed cyclic form or a ring-opened form in DNA, to better understand the mechanisms that Pols iota and kappa employ to promote replication through this lesion, we have examined the ability of these polymerases to replicate through the structural analogs of gamma-HOPdG that are permanently either ring closed or ring opened. Our studies with these model adducts show that whereas the ring-opened form of gamma-HOPdG is not inhibitory to synthesis by human Pols eta, iota, or kappa, only Poliota is able to incorporate nucleotides opposite the ring-closed form, which is known to adopt a syn conformation in DNA. From these studies, we infer that (i) Pols eta, iota, and kappa have the ability to proficiently replicate through minor-groove DNA lesions that do not perturb the Watson-Crick hydrogen bonding of the template base with the incoming nucleotide, and (ii) Poliota can accommodate a minor-groove-adducted template purine which adopts a syn conformation in DNA and forms a Hoogsteen base pair with the incoming nucleotide.     

Activities of human DNA polymerase kappa in response to the major benzo[a]pyrene DNA adduct: error-free lesion bypass and extension synthesis from opposite the lesion

In cells, the major benzo[a]pyrene DNA adduct is the highly mutagenic (+)-trans-anti-BPDE-N(2)-dG. In eukaryotes, little is known about lesion bypass of this DNA adduct during replication. Here, we show that purified human Polkappa can effectively bypass a template (+)-trans-anti-BPDE-N(2)-dG adduct in an error-free manner. Kinetic parameters indicate that Polkappa bypass of the (-)-trans-anti-BPDE-N(2)-dG adduct was approximately 41-fold more efficient compared to the (+)-trans-anti-BPDE-N(2)-dG adduct. Furthermore, we have found another activity of human Polkappa in response to the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts: extension synthesis from mispaired primer 3' ends opposite the lesion. In contrast, the two adducts strongly blocked DNA synthesis by the purified human Polbeta and the purified catalytic subunits of yeast Polalpha, Poldelta, and Pol epsilon right before the lesion. Extension by human Polkappa from the primer 3' G opposite the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts was mediated by a -1 deletion mechanism, probably resulting from re-aligning the primer G to pair with the next template C by Polkappa prior to DNA synthesis. Thus, sequence contexts 5' to the lesion strongly affect the fidelity and mechanism of the Polkappa-catalyzed extension synthesis. These results support a dual-function model of human Polkappa in bypass of BPDE DNA adducts: it may function both as an error-free bypass polymerase alone and an extension synthesis polymerase in combination with another polymerase.     

The spacious active site of a Y-family DNA polymerase facilitates promiscuous nucleotide incorporation opposite a bulky carcinogen-DNA adduct: elucidating the structure-function relationship through experimental and computational approaches

Y-family DNA polymerases lack some of the mechanisms that replicative DNA polymerases employ to ensure fidelity, resulting in higher error rates during replication of undamaged DNA templates and the ability to bypass certain aberrant bases, such as those produced by exposure to carcinogens, including benzo[a]pyrene (BP). A tumorigenic metabolite of BP, (+)-anti-benzo-[a]pyrene diol epoxide, attacks DNA to form the major 10S (+)-trans-anti-[BP]-N(2)-dG adduct, which has been shown to be mutagenic in a number of prokaryotic and eukaryotic systems. The 10S (+)-trans-anti-[BP]-N(2)-dG adduct can cause all three base substitution mutations, and the SOS response in Escherichia coli increases bypass of bulky adducts, suggesting that Y-family DNA polymerases are involved in the bypass of such lesions. Dpo4 belongs to the DinB branch of the Y-family, which also includes E. coli pol IV and eukaryotic pol kappa. We carried out primer extension assays in conjunction with molecular modeling and molecular dynamics studies in order to elucidate the structure-function relationship involved in nucleotide incorporation opposite the bulky 10S (+)-trans-anti-[BP]-N(2)-dG adduct by Dpo4. Dpo4 is able to bypass the 10S (+)-trans-anti-[BP]-N(2)-dG adduct, albeit to a lesser extent than unmodified guanine, and the V(max) values for insertion of all four nucleotides opposite the adduct by Dpo4 are similar. Computational studies suggest that 10S (+)-trans-anti-[BP]-N(2)-dG can be accommodated in the active site of Dpo4 in either the anti or syn conformation due to the limited protein-DNA contacts and the open nature of both the minor and major groove sides of the nascent base pair, which can contribute to the promiscuous nucleotide incorporation opposite this lesion.     

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

1,N(6)-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) alpha, beta, eta and iota. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N(6)-ethenoadenine (epsilonA). Using a primer extension assay, both pols alpha and beta were primarily blocked by EA or epsilonA with very minor extension. Pol eta, a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol eta incorporated all four nucleotides opposite EA and epsilonA, but with differential preferences and mainly in an error-prone manner. Human pol iota, a paralog of human pol eta, was blocked by both adducts with a very small amount of synthesis past epsilonA. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. epsilonA, could affect the specificity of pol iota toward the template T immediately 3' to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or epsilonA showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to epsilonA, is a miscoding lesion.     

Lesion bypass of N2-ethylguanine by human DNA polymerase iota

Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase iota was measured and structures of the DNA polymerase iota-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase iota opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase iota incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase iota opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase iota extends from the N2-ethyl-Gua:Cyt 3' terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase iota catalyzed reaction is well suited for N(2)-ethyl-Gua bypass. The structure of DNA polymerase iota with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase iota complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase iota polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase iota efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.     

Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase kappa

The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Poleta, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Polkappa, which differs from Poleta in having a higher fidelity and which, unlike Poleta, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Poleta. We find that an isosteric thymidine analog is replicated with low efficiency by Polkappa, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Poleta and Polkappa rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.     

Discrimination against the cytosine analog tC by Escherichia coli DNA polymerase IV DinB

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson-Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.     

Discrimination against major groove adducts by Y-family polymerases of the DinB subfamily

Y-family DNA polymerases bypass DNA adducts in a process known as translesion synthesis (TLS). Y-family polymerases make contacts with the minor groove side of the DNA substrate at the nascent base pair. The Y-family polymerases also contact the DNA major groove via the unique little finger domain, but they generally lack contacts with the major groove at the nascent base pair. Escherichia coli DinB efficiently and accurately copies certain minor groove guanosine adducts. In contrast, we previously showed that the presence in the DNA template of the major groove-modified base 1,3-diaza-2-oxophenothiazine (tC) inhibits the activity of E. coli DinB. Even when the DNA primer is extended up to three nucleotides beyond the site of the tC analog, DinB activity is strongly inhibited. These findings prompted us to investigate discrimination against other major groove modifications by DinB and its orthologs. We chose a set of pyrimidines and purines with modifications in the major groove and determined the activity of DinB and several orthologs with these substrates. DinB, human pol kappa, and Sulfolobus solfataricus Dpo4 show differing specificities for the major groove adducts pyrrolo-dC, dP, N⁶-furfuryl-dA, and etheno-dA. In general, DinB was least efficient for bypass of all of these major groove adducts, whereas Dpo4 was most efficient. DinB activity was essentially completely inhibited by the presence of etheno-dA, while pol kappa activity was strongly inhibited. All three of these DNA polymerases were able to bypass N⁶-furfuryl-dA with modest efficiency, with DinB being the least efficient. We also determined that the R35A variant of DinB enhances bypass of N⁶-furfuryl-dA but not etheno-dA. In sum, we find that whereas DinB is specific for bypass of minor groove adducts, it is specifically inhibited by major groove DNA modifications.     

Mechanism of efficient and accurate nucleotide incorporation opposite 7,8-dihydro-8-oxoguanine by Saccharomyces cerevisiae DNA polymerase eta

Most DNA polymerases incorporate nucleotides opposite template 7,8-dihydro-8-oxoguanine (8-oxoG) lesions with reduced efficiency and accuracy. DNA polymerase (Pol) eta, which catalyzes the error-free replication of template thymine-thymine (TT) dimers, has the unique ability to accurately and efficiently incorporate nucleotides opposite 8-oxoG templates. Here we have used pre-steady-state kinetics to examine the mechanisms of correct and incorrect nucleotide incorporation opposite G and 8-oxoG by Saccharomyces cerevisiae Pol eta. We found that Pol eta binds the incoming correct dCTP opposite both G and 8-oxoG with similar affinities, and it incorporates the correct nucleotide bound opposite both G and 8-oxoG with similar rates. While Pol eta incorporates an incorrect A opposite 8-oxoG with lower efficiency than it incorporates a correct C, it does incorporate A more efficiently opposite 8-oxoG than opposite G. This is mainly due to greater binding affinity for the incorrect incoming dATP opposite 8-oxoG. Overall, these results show that Pol eta replicates through 8-oxoG without any barriers introduced by the presence of the lesion.     

Stimulation of DNA Synthesis Activity of Human DNA Polymerase κ by PCNA

Humans have three DNA polymerases, Poleta, Polkappa, and Poliota, which are able to promote replication through DNA lesions. However, the mechanism by which these DNA polymerases are targeted to the replication machinery stalled at a lesion site has remained unknown. Here, we provide evidence for the physical interaction of human Polkappa (hPolkappa) with proliferating cell nuclear antigen (PCNA) and show that PCNA, replication factor C (RFC), and replication protein A (RPA) act cooperatively to stimulate the DNA synthesis activity of hPolkappa. The processivity of hPolkappa, however, is not significantly increased in the presence of these protein factors. The efficiency (V(max)/K(m)) of correct nucleotide incorporation by hPolkappa is enhanced approximately 50- to 200-fold in the presence of PCNA, RFC, and RPA, and this increase in efficiency is achieved by a reduction in the apparent K(m) for the nucleotide. Although in the presence of these protein factors, the efficiency of the insertion of an A nucleotide opposite an abasic site is increased approximately 40-fold, this reaction still remains quite inefficient; thus, it is unlikely that hPolkappa would bypass an abasic site by inserting a nucleotide opposite the site.     

Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases

Deranged oxidative metabolism is a property of many tumour cells. Oxidation of the deoxynucleotide triphosphate (dNTP) pool, as well as DNA, is a major cause of genome instability. Here, we report that two Y-family DNA polymerases of the archaeon Sulfolobus solfataricus strains P1 and P2 incorporate oxidized dNTPs into nascent DNA in an erroneous manner: the polymerases exclusively incorporate 8-OH-dGTP opposite adenine in the template, and incorporate 2-OH-dATP opposite guanine more efficiently than opposite thymine. The rate of extension of the nascent DNA chain following on from these incorporated analogues is only slightly reduced. These DNA polymerases have been shown to bypass a variety of DNA lesions. Thus, our results suggest that the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis, in addition to facilitating translesion DNA synthesis. We also report that human DNA polymerase η, a human Y-family DNA polymerase, incorporates the oxidized dNTPs in a similar erroneous manner.     

Steric and electrostatic effects in DNA synthesis by the SOS-induced DNA polymerases II and IV of Escherichia coli

The SOS-induced DNA polymerases II and IV (pol II and pol IV, respectively) of Escherichia coli play important roles in processing lesions that occur in genomic DNA. Here we study how electrostatic and steric effects play different roles in influencing the efficiency and fidelity of DNA synthesis by these two enzymes. These effects were probed by the use of nonpolar shape analogues of thymidine, in which substituted toluenes replace the polar thymine base. We compared thymine with nonpolar analogues to evaluate the importance of hydrogen bonding in the polymerase active sites, while we used comparisons among a set of variably sized thymine analogues to measure the role of steric effects in the two enzymes. Steady-state kinetics measurements were carried out to evaluate activities for nucleotide insertion and extension. The results showed that both enzymes inserted nucleotides opposite nonpolar template bases with moderate to low efficiency, suggesting that both polymerases benefit from hydrogen bonding or other electrostatic effects involving the template base. Surprisingly, however, pol II inserted nonpolar nucleotide (dNTP) analogues into a primer strand with high (wild-type) efficiency, while pol IV handled them with an extremely low efficiency. Base pair extension studies showed that both enzymes bypass non-hydrogen-bonding template bases with moderately low efficiency, suggesting a possible beneficial role of minor groove hydrogen bonding interactions at the N-1 position. Measurement of the two polymerases' sensitivity to steric size changes showed that both enzymes were relatively flexible, yielding only small kinetic differences with increases or decreases in nucleotide size. Comparisons are made to recent data for DNA pol I (Klenow fragment), the archaeal polymerase Dpo4, and human pol kappa.     

Structure of a high fidelity DNA polymerase bound to a benzo[a]pyrene adduct that blocks replication

Of the carcinogens to which humans are most frequently exposed, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) is one of the most ubiquitous. BP is a byproduct of grilled foods and tobacco and fuel combustion and has long been linked to various human cancers, particularly lung and skin. BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases. Here we present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2-guanine adduct base-paired with cytosine. The BP adduct adopts a conformation that places the polycyclic BP moiety in the nascent DNA minor groove and is the first structure of a minor groove adduct bound to a polymerase. Orientation of the BP moiety into the nascent DNA minor groove results in extensive disruption to the interactions between the adducted DNA duplex and the polymerase. The disruptions revealed by the structure of Bacillus fragment bound to a BP adduct provide a molecular basis for rationalizing the potent blocking effect on replication exerted by BP adducts.     

Translesion synthesis past platinum DNA adducts by human DNA polymerase mu

DNA polymerase mu (pol mu) is a member of the pol X family of DNA polymerases, and it shares a number of characteristics of both DNA polymerase beta (pol beta) and terminal deoxynucleotidyl transferase (TdT). Because pol beta has been shown to perform translesion DNA synthesis past cisplatin (CP)- and oxaliplatin (OX)-GG adducts, we determined the ability of pol mu to bypass these lesions. Pol mu bypassed CP and OX adducts with an efficiency of 14-35% compared to chain elongation on undamaged DNA, which is second only to pol eta in terms of bypass efficiency. The relative ability of pol mu to bypass CP and OX adducts was dependent on both template structure and sequence context. Since pol mu has been shown to be more efficient on gapped DNA templates than on primed single-stranded DNA templates, we determined the ability of pol mu to bypass Pt-DNA adducts on both primed single-stranded and gapped templates. The bypass of Pt-DNA adducts by pol mu was highly error-prone on all templates, resulting in 2, 3, and 4 nt deletions. We postulate that bypass of Pt-DNA adducts by pol mu may involve looping out the Pt-GG adduct to allow chain elongation downstream of the adduct. This reaction appears to be facilitated by the presence of a downstream "acceptor" and a gap large enough to provide undamaged template DNA for elongation past the adduct, although gapped DNA is clearly not required for bypass.     

Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases beta and eta

DNA polymerases beta (pol beta ) and eta (pol eta ) are the only two eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Frameshift errors are an important aspect of mutagenesis. We have compared the types of frameshifts that occur during translesion synthesis past cisplatin and oxaliplatin adducts in vitro by pol beta and pol eta on a template containing multiple runs of nucleotides flanking a single platinum-GG adduct. Translesion synthesis past platinum adducts by pol beta resulted in approximately 50% replication products containing single-base deletions. For both adducts the majority of -1 frameshifts occurred in a TTT sequence 3-5 bp upstream of the DNA lesion. For pol eta, all of the bypass products for both cisplatin and oxaliplatin adducts contained -1 frameshifts in the upstream TTT sequence and most of the products of replication on oxaliplatin-damaged templates had multiple replication errors, both frameshifts and misinsertions. In addition, on platinated templates both polymerases generated replication products 4-8 bp shorter than the full-length products. The majority of short cisplatin-induced products contained an internal deletion which included the adduct. In contrast, the majority of oxaliplatin-induced short products contained a 3' terminal deletion. The implications of these in vitro results for in vivo mutagenesis are discussed.     

Mutagenic nucleotide incorporation and hindered translocation by a food carcinogen C8-dG adduct in Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): modeling and dynamics studies

Bulky carcinogen-DNA adducts commonly cause replicative polymerases to stall, leading to a switch to bypass polymerases. We have investigated nucleotide incorporation opposite the major adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the DinB family polymerase, Dpo4, using molecular modeling and molecular dynamics (MD) simulations. PhIP, the most prevalent heterocyclic aromatic amine formed by cooking of proteinaceous food, is mutagenic in mammalian cells and is implicated in mammary and colon tumors. Our results show that the dG-C8-PhIP adduct can be accommodated in the spacious major groove Dpo4 open pocket, with Dpo4 capable of incorporating dCTP, dTTP or dATP opposite the adduct reasonably well. However, the PhIP ring system on the minor groove side would seriously disturb the active site, regardless of the presence and identity of dNTP. Furthermore, the simulations indicate that dATP and dTTP are better incorporated in the damaged system than in their respective mismatched but unmodified controls, suggesting that the PhIP adduct enhances incorporation of these mismatches. Finally, bulky C8-dG adducts, situated in the major groove, are likely to impede translocation in this polymerase (Rechkoblit et al. (2006), PLoS Biol., 4, e11). However, N²-dG adducts, which can reside on the minor groove side, appear to cause less hindrance when in this position.     

trans-Lesion synthesis past bulky benzo[a]pyrene diol epoxide N2-dG and N6-dA lesions catalyzed by DNA bypass polymerases

The effectiveness of in vitro primer elongation reactions catalyzed by human bypass DNA polymerases kappa (hDinB1), pol eta (hRad30A), pol iota (hRad30B), and yeast pol zeta (Rev3 and Rev7) in site-specifically modified template oligonucleotide strands were studied in vitro. The templates contained single bulky lesions derived from the trans-addition of the mutagenic (+)- or (-)-enantiomers of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (a metabolite of the environmental carcinogen benzo[a]pyrene), to the exocyclic amino groups of guanine or adenine in oligonucleotide templates 33, or more, bases long. In "running start" primer extension reactions, pol kappa effectively bypassed both the stereoisomeric (+)- and (-)-trans-guanine adducts but not the analogous adenine adducts. In sharp contrast, pol eta, which exhibits considerable sequence homology with pol kappa (both belong to the group of Y family polymerases), is partially blocked by the guanine adducts and the (-)-trans-adenine adduct, although the stereoisomeric (+)-trans-adenine adduct is more successfully bypassed. Neither pol iota nor pol zeta, either alone or in combination, were effective in trans-lesion synthesis past the same adducts. In all cases, the fidelity of insertion is dependent on adduct stereochemistry and structure. Generally, error-free nucleotide insertion opposite the lesions tends to depend more on adduct stereochemistry than error-prone insertion. None of the polymerases tested are a universal bypass polymerase for the stereoisomeric bulky polycyclic aromatic hydrocarbon-DNA adducts derived from anti-BPDE.     

Translesion synthesis past acrolein-derived DNA adduct,gamma -hydroxypropanodeoxyguanosine,by yeast and human DNA polymerase eta

gamma-Hydroxy-1,N(2)-propano-2'deoxyguanosine (gamma-HOPdG) is a major deoxyguanosine adduct derived from acrolein, a known mutagen. In vitro, this adduct has previously been shown to pose a severe block to translesion synthesis by a number of polymerases (pol). Here we show that both yeast and human pol eta can incorporate a C opposite gamma-HOPdG at approximately 190- and approximately 100-fold lower efficiency relative to the control deoxyguanosine and extend from a C paired with the adduct at approximately 8- and approximately 19-fold lower efficiency. Although DNA synthesis past gamma-HOPdG by yeast pol eta was relatively accurate, the human enzyme misincorporated nucleotides opposite the lesion with frequencies of approximately 10(-1) to 10(-2). Because gamma-HOPdG can adopt both ring closed and ring opened conformations, comparative replicative bypass studies were also performed with two model adducts, propanodeoxyguanosine and reduced gamma-HOPdG. For both yeast and human pol eta, the ring open reduced gamma-HOPdG adduct was less blocking than gamma-HOPdG, whereas the ring closed propanodeoxyguanosine adduct was a very strong block. Replication of DNAs containing gamma-HOPdG in wild type and xeroderma pigmentosum variant cells revealed a somewhat decreased mutation frequency in xeroderma pigmentosum variant cells. Collectively, the data suggest that pol eta might potentially contribute to both error-free and mutagenic bypass of gamma-HOPdG.