Translesion synthesis past tamoxifen-derived DNA adducts by human DNA polymerases eta and kappa

The long-term treatment of tamoxifen (TAM), widely used for adjuvant chemotherapy and chemoprevention for breast cancer, increases a risk of developing endometrial cancer. A high frequency of K-ras mutations has been observed in the endometrium of women treated with TAM. Human DNA polymerase (pol) eta and pol kappa are highly expressed in the reproductive organs and are associated with translesion synthesis past bulky DNA adducts. To explore the miscoding properties of alpha-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM), a major TAM-DNA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of trans or cis forms of dG-N2-TAM were prepared by phosphoramidite chemical procedure and used as templates. The primer extension reaction catalyzed by pol kappa deltaC, a truncated form of pol kappa, extended more efficiently past the adduct than that of pol eta by incorporating dCMP, a correct base, opposite the adduct. With pol eta, all diastereoisomers of dG-N2-TAM promoted small amounts of direct incorporation of dAMP and deletions. With pol kappa deltaC, dG-N2-TAM promoted small amounts of dTMP and/or dAMP incorporations and deletions. The miscoding properties varied depending on the diastereoisomer of dG-N2-TAM adducts and the DNA pol used. Steady-state kinetic studies were also performed using either the nonspecific sequence or the K-ras gene sequence containing a single dG-N2-TAM at the second base of codon 12. With pol eta, the bypass frequency past the dA x dG-N2-TAM pair positioned in the K-ras sequence was only 2.3 times lower than that for the dC x dG-N2-TAM pair, indicating that dG-N2-TAM in the K-ras sequence has higher miscoding potential than that in the nonspecific sequence. However, with pol kappa deltaC, the bypass frequency past the dC x dG-N2-TAM pair was higher than that of the dT x dG-N2-TAM pair in both sequences. The properties of pol eta and pol kappa are consistent with the mutagenic events attributed to TAM-DNA adducts.     

Translesion synthesis past estrogen-derived DNA adducts by human DNA polymerases eta and kappa

Newly discovered human DNA polymerase (pol) eta and kappa are highly expressed in the reproductive organs, such as testis, ovary, and uterus, where steroid hormones are produced. Because treatment with estrogen increases the risk of developing breast, ovary, and endometrial cancers, miscoding events occurring at model estrogen-derived DNA adducts were explored using pol eta and a truncated form of human pol kappa (pol kappaDeltaC). These enzymes bypassed N(2)-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'-deoxyguanosine (dG-N(2)-3MeE) and N(6)-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'-deoxyadenosine (dA-N(6)-3MeE), which were embedded in site-specifically modified oligodeoxynucleotide templates. Quantitative analysis of base substitutions and deletions occurring at the lesion site showed that pol kappaDeltaC was more efficient at incorporating dCMP opposite the dG-N(2)-3MeE lesion than pol eta. Surprisingly, the frequency of translesion synthesis beyond the dC*dG-N(2)-3MeE pair was 13% of the normal dC*dG pair and was 4 and 6 orders of magnitude higher than that of dC*(+)-trans-dG-N(2)-benzo[a]pyrene and dC*dG-C8-acetylaminofluorene pairs, respectively, suggesting that dG-N(2)-3MeE is a natural substrate for pol kappa. In contrast, the bypass frequency beyond the dT*dA-N(6)-3MeE pair was 7 orders of magnitude less than that for the normal dT*dA pair. dA-N(6)-3MeE is a more miscoding lesion than dG-N(2)-3MeE. Pol eta promoted incorporation of dAMP and dCMP at the dA-N(6)-3MeE lesion, while with pol kappaDeltaC, deletions were more frequently observed, along with incorporation of dAMP and dCMP opposite the lesion. These observations were also supported by steady-state kinetic studies. When taken together, the properties of pol eta and kappa are consistent with the mutagenic events attributed to estrogen-derived DNA adducts.     

Translesion synthesis past equine estrogen-derived 2'-deoxyadenosine DNA adducts by human DNA polymerases eta and kappa

Hormone replacement therapy (HRT) increases the risk of developing breast, ovarian, and endometrial cancers. Equilin and equilenin are the major components of the widely prescribed drug used for HRT. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equilin and equilenin, promotes 4-OHEN-modified dC, dA, and dG DNA adducts. These DNA adducts were detected in breast tumor and adjacent normal tissues of several patients receiving HRT. We have recently found that the 4-OHEN-dC DNA adduct is a highly miscoding lesion generating C --> T transitions and C --> G transversions. To explore the mutagenic potential of another major 4-OHEN-dA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of 4-OHEN-dA (Pk-1, Pk-2, and Pk-3) were prepared by a postsynthetic method and used as DNA templates for primer extension reactions catalyzed by human DNA polymerase (pol) eta and kappa that are highly expressed in the reproductive organs. Primer extension catalyzed by pol eta or pol kappa occurred rapidly on the unmodified template to form fully extended products. With the major 4-OHEN-dA-modified templates (Pk-2 and Pk-3), primer extension was retarded prior to the lesion and opposite the lesion; a fraction of the primers was extended past the lesion. Steady-state kinetic studies with pol eta and pol kappa indicated that dTMP, the correct base, was preferentially incorporated opposite the 4-OHEN-dA lesion. In addition, pol eta and pol kappa bypassed the lesion by incorporating dAMP and dCMP, respectively, opposite the lesion and extended past the lesion. The relative bypass frequency past the 4-OHEN-dA lesion with pol eta was at least 2 orders of magnitude higher than that observed with pol kappa. The bypass frequency past Pk-2 was more efficient than that past Pk-3. Thus, 4-OHEN-dA is a miscoding lesion generating A --> T transversions and A --> G transitions. The miscoding frequency and specificity of 4-OHEN-dA varied depending on the stereoisomer of the 4-OHEN-dA adduct and DNA polymerase used.     

Translesion synthesis past equine estrogen-derived 2'-deoxycytidine DNA adducts by human DNA polymerases eta and kappa

Estrogen replacement therapy (ERT), composed of equilenin, is associated with increased risk of breast, ovarian, and endometrial cancers. Several diastereoisomers of unique dC and dA DNA adducts were derived from 4-hydroxyequilenin (4-OHEN), a metabolite of equilenin, and have been detected in women receiving ERT. To explore the miscoding property of 4-OHEN-dC adduct, site-specifically modified oligodeoxynucleotides (Pk-1, Pk-2, Pk-3, and Pk-4) containing a single diastereoisomer of 4-OHEN-dC were prepared by a postsynthetic method. Among them, major 4-OHEN-dC-modified oligodeoxynucleotides (Pk-3 and Pk-4) were used to prepare the templates for primer extension reactions catalyzed by DNA polymerase (pol) alpha, pol eta, and pol kappa. Primer extension was retarded one base prior to the lesion and opposite the lesion; stronger blockage was observed with pol alpha, while with human pol eta or pol kappa, a fraction of the primers was extended past the lesion. Steady-state kinetic studies showed that both pol kappa and pol eta inserted dCMP and dAMP opposite the 4-OHEN-dC and extended past the lesion. Never or less-frequently, dGMP, the correct base, was inserted opposite the lesion. The relative bypass frequency past the 4-OHEN-dC lesion with pol eta was at least 3 orders of magnitude higher than that for pol kappa, as observed for primer extension reactions. The bypass frequency past the dA.4-OHEN-dC adduct in Pk-4 was 2 orders of magnitude more efficient than that past the adduct in Pk-3. Thus, 4-OHEN-dC is a highly miscoding lesion capable of generating C --> T transitions and C --> G transversions. The miscoding frequency and specificity of 4-OHEN-dC were strikingly influenced by the adduct stereochemistry and DNA polymerase used.     

Two-step error-prone bypass of the (+)- and (−)-trans-anti-BPDE-N-dG adducts by human DNA polymerases η and κ

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase η (Polη) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Polη predominantly inserted an A opposite a template (+)- and (−)-trans-anti-BPDE-N²-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Polη. Error-prone nucleotide insertion by human Polη was more efficient opposite the (+)-trans-anti-BPDE-N²-dG adduct than opposite the (−)-trans-anti-BPDE-N²-dG. However, translesion synthesis by human Polη largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Polη from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N²-dG adducts, the nucleotide opposite the lesion, and the sequence context 5′ to the lesion. By combining the nucleotide insertion activity of human Polη and the extension synthesis activity of human Polκ, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (−)-trans-anti-BPDE-N²-dG DNA adducts.     

Mutagenic bypass of the butadiene-derived 2'-deoxyuridine adducts by polymerases eta and zeta

Butadiene is a ubiquitous environmental chemical carcinogen that when activated to its monoepoxide intermediate can react with the N3 position of cytosine, resulting in two stereoisomeric adducted bases that rapidly deaminate to N3 2′-deoxyuridine lesions. We have previously shown that replication of DNAs containing these adducts through mammalian cells resulted in 97% mutagenicity, predominantly C to T transitions. Since replicative DNA polymerases were blocked by these lesions in vitro, translesional polymerases were assessed for their ability to bypass these adducts. While polymerases ι, κ and ζ were significantly blocked one nucleotide prior to the lesion, pol η incorporated nucleotides opposite the adducts with a preference for insertion of a G or A. Following polymerase dissociation and reassociation, pol η was also able to extend primers with mispaired termini opposite the lesions, with extensions from the A and T mismatched primer termini being the most efficient. Pol ζ was also able to extend primers containing all mismatched nucleotides opposite the lesions, with the most efficient extension occurring off of the A mismatched primer.     

Poleta, Polzeta and Rev1 together are required for G to T transversion mutations induced by the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts in yeast cells

Benzo[a]pyrene is an important environmental mutagen and carcinogen. Its metabolism in cells yields the mutagenic, key ultimate carcinogen 7R,8S,9S,10R-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, (+)-anti-BPDE, which reacts via its 10-position with N²-dG in DNA to form the adduct (+)-trans-anti-BPDE-N²-dG. To gain molecular insights into BPDE-induced mutagenesis, we examined in vivo translesion synthesis and mutagenesis in yeast cells of a site-specific 10S (+)-trans-anti-BPDE-N²-dG adduct and the stereoisomeric 10R (−)-trans-anti-BPDE-N²-dG adduct. In wild-type cells, bypass products consisted of 76% C, 14% A and 7% G insertions opposite (+)-trans-anti-BPDE-N²-dG; and 89% C, 4% A and 4% G insertions opposite (−)-trans-anti-BPDE-N²-dG. Translesion synthesis was reduced by ~26–37% in rad30 mutant cells lacking Polη, but more deficient in rev1 and almost totally deficient in rev3 (lacking Polζ) mutants. C insertion opposite the lesion was reduced by ~24–33% in rad30 mutant cells, further reduced in rev1 mutant, and mostly disappeared in the rev3 mutant strain. The insertion of A was largely abolished in cells lacking either Polη, Polζ or Rev1. The insertion of G was not detected in either rev1 or rev3 mutant cells. The rad30 rev3 double mutant exhibited a similar phenotype as the single rev3 mutant with respect to translesion synthesis and mutagenesis. These results show that while the Polζ pathway is generally required for translesion synthesis and mutagenesis of the (+)- and (−)-trans-anti-BPDE-N²-dG DNA adducts, Polη, Polζ and Rev1 together are required for G→T transversion mutations, a major type of mutagenesis induced by these lesions. Based on biochemical and genetic results, we present mechanistic models of translesion synthesis of these two DNA adducts, involving both the one-polymerase one-step and two-polymerase two-step models.     

Error-free and error-prone lesion bypass by human DNA polymerase kappa in vitro

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polκ encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polκ is a novel lesion bypass polymerase in vitro. Purified human Polκ efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polκ most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polκ was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polκ effectively bypassed a template (–)-trans-anti-benzo[a]pyrene-N²-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polκ was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polκ plays an important role in both error-free and error-prone lesion bypass in humans.     

Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase iota

DNA polymerase (pol) iota has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol iota. Pol iota effectively bypassed N2-methyl (Me)G and N2-ethyl(Et)G, partially bypassed N2-isobutyl(Ib)G and N2-benzylG, and was blocked at N2-CH2(2-naphthyl)G (N2-NaphG), N2-CH2(9-anthracenyl)G (N2-AnthG), and N2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of kcat/Km for dCTP insertion opposite N2-G adducts according to size, with a maximal decrease opposite N2-AnthG (61-fold). dTTP misinsertion frequency opposite template G was increased 3-11-fold opposite adducts (highest with N2-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N2-IbG, N2-NaphG, and N2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (S(p)-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N2-EtG and N2-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N2,N2-diMeG compared with N2-EtG by pol eta but not by pol iota is consistent with Hoogsteen base pairing by pol iota. Thus, polymerization by pol iota is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol iota may play a limited role in translesion synthesis on bulky N2-G adducts in cells.     

Functions of human DNA polymerases eta,kappa and iota suggested by their properties,including fidelity with undamaged DNA templates

Human DNA polymerases eta, kappa and iota are template-dependent, Y-family DNA polymerases that have been implicated in translesion DNA synthesis (TLS) in human cells. Here, we briefly review evidence that these exonuclease-deficient polymerases copy undamaged DNA with very low fidelity and unusual error specificity. Based on the base substitution specificity and other biochemical properties of DNA polymerases eta and iota, we consider the possibility that they participate in specialized DNA transactions that repair damaged DNA and/or generate mutations in the variable regions of immunoglobulin genes.     

Error-prone lesion bypass by human DNA polymerase eta

DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase η (Polη), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Polη, we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Polη efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Polη effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant –1 deletion was also observed when the template base 5′ to the abasic site is a T. Human Polη partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N²-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N²-dG lesion in mammalian cells. These results show that human Polη is capable of error-prone translesion DNA syntheses in vitro and suggest that Polη may bypass certain lesions with a mutagenic consequence in humans.     

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.     

The Werner syndrome protein limits the error-prone 8-oxo-dG lesion bypass activity of human DNA polymerase kappa

Human DNA polymerase kappa (hpol κ) is the only Y-family member to preferentially insert dAMP opposite 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) during translesion DNA synthesis. We have studied the mechanism of action by which hpol κ activity is modulated by the Werner syndrome protein (WRN), a RecQ helicase known to influence repair of 8-oxo-dG. Here we show that WRN stimulates the 8-oxo-dG bypass activity of hpol κ in vitro by enhancing the correct base insertion opposite the lesion, as well as extension from dC:8-oxo-dG base pairs. Steady-state kinetic analysis reveals that WRN improves hpol κ-catalyzed dCMP insertion opposite 8-oxo-dG ∼10-fold and extension from dC:8-oxo-dG by 2.4-fold. Stimulation is primarily due to an increase in the rate constant for polymerization (k_pol), as assessed by pre-steady-state kinetics, and it requires the RecQ C-terminal (RQC) domain. In support of the functional data, recombinant WRN and hpol κ were found to physically interact through the exo and RQC domains of WRN, and co-localization of WRN and hpol κ was observed in human cells treated with hydrogen peroxide. Thus, WRN limits the error-prone bypass of 8-oxo-dG by hpol κ, which could influence the sensitivity to oxidative damage that has previously been observed for Werner''s syndrome cells.     

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.     

Translesion synthesis across bulky N2-alkyl guanine DNA adducts by human DNA polymerase kappa

DNA polymerase (pol) kappa is one of the so-called translesion polymerases involved in replication past DNA lesions. Bypass events have been studied with a number of chemical modifications with human pol kappa, and the conclusion has been presented, based on limited quantitative data, that the enzyme is ineffective at incorporating opposite DNA damage but proficient at extending beyond bases paired with the damage. Purified recombinant full-length human pol kappa was studied with a series of eight N(2)-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH(2)(6-benzo[a]pyrenyl) (BP). Steady-state kinetic parameters (catalytic specificity, k(cat)/K(m)) were similar for insertion of dCTP opposite the lesions and for extension beyond the N(2)-adduct G:C pairs. Mispairing of dGTP and dTTP was similar and occurred with k(cat)/K(m) values approximately 10(-3) less than for dCTP with all adducts; a similar differential was found for extension beyond a paired adduct. Pre-steady-state kinetic analysis showed moderately rapid burst kinetics for dCTP incorporations, even opposite the bulky methyl(9-anthracenyl)- and BPG adducts (k(p) 5.9-10.3 s(-1)). The rapid bursts were abolished opposite BPG when alpha-thio-dCTP was used instead of dCTP, implying rate-limiting phosphodiester bond formation. Comparisons are made with similar studies done with human pols eta and iota; pol kappa is the most resistant to N(2)-bulk and the most quantitatively efficient of these in catalyzing dCTP incorporation opposite bulky guanine N(2)-adducts, particularly the largest (N(2)-BPG).     

Efficiency of extension of mismatched primer termini across from cisplatin and oxaliplatin adducts by human DNA polymerases beta and eta in vitro

DNA polymerases beta and eta are among the few eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Our laboratory has previously established that both polymerases misincorporated dTTP with high frequency across from cisplatin- and oxaliplatin-GG adducts. This decrease in polymerase fidelity on platinum-damaged DNA could lead to in vivo mutations, if this base substitution were efficiently elongated. In this study, we performed a steady-state kinetic analysis of the steps required for fixation of dTTP misinsertion during translesion synthesis past cisplatin- and oxaliplatin-GG adducts by pol beta and pol eta. The efficiency of translesion synthesis by pol eta past Pt-GG adducts was very similar to that observed for this polymerase when the template contains thymine-thymine dimers. This finding suggested that pol eta could play a role in translesion synthesis past platinum-GG adducts in vivo. On the other hand, translesion synthesis past platinum-GG adducts by pol beta was much less efficient. Translesion synthesis by pol eta is likely to be predominantly error-free, since the probability of correct insertion and extension by pol eta was 1000-2000-fold greater than the probability of incorrect insertion and extension. Our results also indicated that for pol eta the frequency of misincorporation is the same across from the 3'G and the 5'G of the platinum-GG adducts for both cisplatin and oxaliplatin adducts. On the other hand, pol beta is more likely to misinsert at the 3'G of the adducts and misinsertion occurs at higher frequency for oxaliplatin-GG than for cisplatin-GG adducts.     

Translesion synthesis across O6-alkylguanine DNA adducts by recombinant human DNA polymerases

Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O(6)-methyl and -benzyl (Bz) G. Recombinant human polymerase (pol) delta also copied past these two lesions but was totally blocked by O(6)-[4-oxo-4-(3-pyridyl)butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. The human translesion pols iota and kappa produced mainly only 1-base incorporation opposite O(6)-MeG and O(6)-BzG and had very low activity in copying O(6)-PobG. Human pol eta copied past all three adducts. Steady-state kinetic analysis showed similar efficiencies of insertion opposite the O(6)-alkylG adducts for dCTP and dTTP with pol eta and kappa; pol iota showed a strong preference for dTTP. pol eta, iota, and kappa showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O(6)-MeG but little, if any, for O(6)-BzG or O(6)-PobG. Analysis of the pol eta O(6)-PobG products indicated that the insertion of G was opposite the base (C) 5' of the adduct, but this product was not extended. Mass spectrometry analysis of all of the pol eta primer extension products indicated multiple components, mainly with C or T inserted opposite O(6)-alkylG but with no deletions in the cases of O(6)-MeG and O(6)-PobG. With pol eta and O(6)-BzG, products were also obtained with -1 and -2 deletions and also with A inserted (opposite O(6)-BzG). The results with pol eta may be relevant to some mutations previously reported with O(6)-alkylG adducts in mammalian cells.     

DNA polymerases eta and kappa exchange with the polymerase delta holoenzyme to complete common fragile site synthesis

Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA.     

Monofunctional DNA-platinum(II) adducts block frequently DNA polymerases.

The question of whether monofunctional DNA platinum(II) adducts block synthesis of DNA by purified DNA polymerases of different types and origin has been investigated by comparing the time dependence of synthesis arrest and of DNA adduct formation. Activated salmon testis DNA is used as a suitable substrate for DNA synthesis allowing to probe inhibition by platinum(II) monoadducts for the variety of inherent template-primers. Reaction amplitudes are related to defined mixtures of dichloro and chloroaqua platinum(II) complexes. It is found that (i) all investigated DNA polymerases seem arrested (100% efficiency) at bifunctional DNA adducts. (ii) human DNA polymerase beta bypasses most of the monofunctional lesions of the three platinum(II) complexes investigated. (iii) Klenow fragment is blocked by monoadducts with increasing efficiency in the order cis-diamminechloroaquaplatinum(II) (0%) less than meso-[1,2-bis(2,6- dichloro-4-hydroxyphenyl)ethylenediamine] chloroaquaplatinum(II) (50%) less than trans-diamminechloro-aquaplatinum(II) (75%). (iv) Escherichia coli DNA polymerase I, Thermus aquaticus DNA polymerase, Physarum polycephalum DNA polymerase alpha, and calf thymus DNA polymerase alpha appear to be arrested by monoadducts. According to these examples, blocking efficiencies depend on the cis/trans-stereogeometry of fixation of the carrier ligands at platinum(II) residues, on the size/chemical nature of the platin(II) carrier ligand and on the type/origin of DNA polymerase.     

Miscoding properties of 6alpha- and 6beta-diastereoisomers of the N(2)-(estradiol-6-yl)-2'-deoxyguanosine DNA adduct by Y-family human DNA polymerases

Treatment with estrogen increases the risk of breast, ovary, and endometrial cancers in women. DNA damage induced by estrogen is thought to be involved in estrogen carcinogenesis. In fact, Y-family human DNA polymerases (pol) eta and kappa, which are highly expressed in the reproductive organs, miscode model estrogen-derived DNA adducts during DNA synthesis. Since the estrogen-DNA adducts are a mixture of 6alpha- and 6beta-diastereoisomers of dG-N(2)-6-estrogen or dA-N(6)-6-estrogen, the stereochemistry of each isomeric adduct on translesion synthesis catalyzed by DNA pols has not been investigated. We have recently established a phosphoramidite chemical procedure to insert 6alpha- or 6beta-isomeric N(2)-(estradiol-6-yl)-2'-deoxyguanosine (dG-N(2)-6-E(2)) into oligodeoxynucleotides. Using such site-specific modified oligomer as a template, the specificity and frequency of miscoding by dG-N(2)-6alpha-E(2) or dG-N(2)-6beta-E(2) were explored using pol eta and a truncated form of pol kappa (pol kappaDeltaC). Translesion synthesis catalyzed by pol eta bypassed both the 6alpha- and 6beta-isomers of dG-N(2)-6-E(2), with a weak blockage at the adduct site, while translesion synthesis catalyzed by pol kappaDeltaC readily bypassed both isomeric adducts. Quantitative analysis of base substitutions and deletions occurring at the adduct site showed that pol kappaDeltaC was more efficient than pol eta by incorporating dCMP opposite both 6alpha- and 6beta-isomeric dG-N(2)-6-E(2) adducts. The miscoding events occurred more frequently with pol eta, but not with pol kappaDeltaC. Pol eta promoted incorporation of dAMP and dTMP at both the 6alpha- and 6beta-isomeric adducts, generating G --> T transversions and G --> A transitions. One- and two-base deletions were also formed. The 6alpha-isomeric adduct promoted slightly lower frequency of dCMP incorporation and higher frequency of dTMP incorporation and one-base deletions, compared with the 6beta-isomeric adduct. These observations were supported by steady-state kinetic studies. Taken together, the miscoding property of the 6alpha-isomeric dG-N(2)-6-E(2) is likely to be similar to that of the 6beta-isomeric adduct.