Replication of damaged DNA in mammalian cells: new solutions to an old problem

All cells need not only to remove damage from their DNA, but also to be able to replicate DNA containing unrepaired damage. In mammalian cells, the major process by which cells are able to replicate damaged templates is translesion synthesis, the direct synthesis of DNA past altered bases. Crucial to this process is a series of recently discovered DNA polymerases. Most of them belong to a new family of polymerases designated the Y-family, which have conserved sequences in the catalytic N-terminal half of the proteins. These polymerases have different efficiencies and specificities in vitro depending on the type of damage in the template.One of them, DNA polymerase eta, is defective in xeroderma pigmentosum variants, and overwhelming evidence suggests that this is the polymerase that carries out translesion synthesis past UV-induced cyclobutane pyrimidine dimers in vivo. DNA polymerase eta is localised in replication factories during DNA replication and accumulates at sites of stalled replication forks. Many studies have been carried out on the properties of the other polymerases in vitro, but there is as yet very little evidence for their specific roles in vivo.     

Proliferating cell nuclear antigen-dependent coordination of the biological functions of human DNA polymerase iota

Y-family DNA polymerases are believed to facilitate the replicative bypass of damaged DNA in a process commonly referred to as translesion synthesis. With the exception of DNA polymerase eta (poleta), which is defective in humans with the Xeroderma pigmentosum variant (XP-V) phenotype, little is known about the cellular function(s) of the remaining human Y-family DNA polymerases. We report here that an interaction between human DNA polymerase iota (poliota) and the proliferating cell nuclear antigen (PCNA) stimulates the processivity of poliota in a template-dependent manner in vitro. Mutations in one of the putative PCNA-binding motifs (PIP box) of poliota or the interdomain connector loop of PCNA diminish the binding between poliota and PCNA and concomitantly reduce PCNA-dependent stimulation of poliota activity. Furthermore, although retaining its capacity to interact with poleta in vivo, the poliota-PIP box mutant fails to accumulate in replication foci. Thus, PCNA, acting as both a scaffold and a modulator of the different activities involved in replication, appears to recruit and coordinate replicative and translesion DNA synthesis polymerases to ensure genome integrity.     

Eukaryotic DNA polymerases, a growing family

In eukaryotic cells, DNA polymerases are required to maintain the integrity of the genome during processes, such as DNA replication, various DNA repair events, translesion DNA synthesis, DNA recombination, and also in regulatory events, such as cell cycle control and DNA damage checkpoint function. In the last two years, the number of known DNA polymerases has increased to at least nine (called alpha, beta, gamma, delta, epsilon, zeta, eta, t and iota), and yeast Saccharomyces cerevisiae contains REV1 deoxycytidyl transferase.     

Xeroderma pigmentosum variant and error-prone DNA polymerases

Replicative DNA synthesis is a faithful event which requires undamaged DNA and high fidelity DNA polymerases. If unrepaired damage remains in the template DNA during replication, specialised low fidelity DNA polymerases synthesises DNA past lesions (translesion synthesis, TLS). Current evidence suggests that the polymerase switch from replicative to translesion polymerases might be mediated by post-translational modifications involving ubiquitination processes. One of these TLS polymerases, polymerase eta carries out TLS past UV photoproducts and is deficient in the variant form of xeroderma pigmentosum (XP-V). The dramatic proneness to skin cancer of XP-V individuals highlights the importance of this DNA polymerase in cancer avoidance. The UV hypermutability of XP-V cells suggests that, in the absence of a functional poleta, UV-induced lesions are bypassed by inaccurate DNA polymerase(s) which remain to be identified.     

Efficiency, fidelity and enzymatic switching during translesion DNA synthesis

More than half of the 16 human DNA polymerases may have some role in DNA replication and potentially modulate the biological effects of DNA template lesions that impede replication fork progression. As one approach to understand how multiple polymerases are coordinated at the fork, we recently quantified the efficiency and fidelity with which one particular translesion synthesis enzyme, human DNA polymerase eta, copies templates containing cis-syn thymine dimers. Several observations from that study were unanticipated. Here we discuss the structural and biological implications of those results in light of earlier studies of translesion synthesis.     

Efficient translesion replication past oxaliplatin and cisplatin GpG adducts by human DNA polymerase eta

Platinum anticancer agents form bulky DNA adducts which are thought to exert their cytotoxic effect by blocking DNA replication. Translesion synthesis, one of the pathways of postreplication repair, is thought to account for some resistance to DNA damage and much of the mutagenicity of bulky DNA adducts in dividing cells. Oxaliplatin has been shown to be effective in cisplatin-resistant cell lines and less mutagenic than cisplatin in the Ames assay. We have shown that the eukaryotic DNA polymerases yeast pol zeta, human pol beta, and human pol gamma bypass oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. Human pol eta, a product of the XPV gene, has been shown to catalyze efficient translesion synthesis past cis, syn-cyclobutane pyrimidine dimers. In the present study we compared translesion synthesis past different Pt-GG adducts by human pol eta. Our data show that, similar to other eukaryotic DNA polymerases, pol eta bypasses oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. However, pol eta-catalyzed translesion replication past Pt-DNA adducts was more efficient and less accurate than that seen for previously tested polymerases. We show that the efficiency and fidelity of translesion replication past Pt-DNA adducts appear to be determined by both the structure of the adduct and the DNA polymerase active site.     

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.     

Co-localization in replication foci and interaction of human Y-family members, DNA polymerase pol eta and REVl protein

The progress of replicative DNA polymerases along the replication fork may be impeded by the presence of lesions in the genome. One way to circumvent such hurdles involves the recruitment of specialized DNA polymerases that perform limited incorporation of nucleotides in the vicinity of the damaged site. This process entails DNA polymerase switch between replicative and specialized DNA polymerases. Five eukaryotic proteins can carry out translesion synthesis (TLS) of damaged DNA in vitro, DNA polymerases zeta, eta, iota, and kappa, and REV1. To identify novel proteins that interact with hpol eta, we performed a yeast two-hybrid screen. In this paper, we show that hREV1 interacts with hpol eta as well as with hpol kappa and poorly with hpol iota. Furthermore, cellular localization analysis demonstrates that hREV1 is present, with hpol eta in replication factories at stalled replication forks and is tightly associated with nuclear structures. This hREV1 nuclear localization occurs independently of the presence of hpol eta. Taken together, our data suggest a central role for hREV1 as a scaffold that recruits DNA polymerases involved in TLS.     

Translesion DNA synthesis catalyzed by human pol eta and pol kappa across 1,N6-ethenodeoxyadenosine

1,N(6)-Ethenodeoxyadenosine, a DNA adduct generated by exogenous and endogenous sources, severely blocks DNA synthesis and induces miscoding events in human cells. To probe the mechanism for in vivo translesion DNA synthesis across this adduct, in vitro primer extension studies were conducted using newly identified human DNA polymerases (pol) eta and kappa, which have been shown to catalyze translesion DNA synthesis past several DNA lesions. Steady-state kinetic analyses and analysis of translesion products have revealed that the synthesis is >100-fold more efficient with pol eta than with pol kappa and that both error-free and error-prone syntheses are observed with these enzymes. The miscoding events include both base substitution and frameshift mutations. These results suggest that both polymerases, particularly pol eta, may contribute to the translesion DNA synthesis events observed for 1,N(6)-ethenodeoxyadenosine in human cells.     

Replication of damaged DNA by translesion synthesis in human cells

Most types of DNA damage block the passage of the replication machinery. In order to bypass these blocks, cells employ special translesion synthesis (TLS) DNA polymerases, which have lower stringency than replicative polymerases. DNA polymerase eta is the major polymerase responsible for bypassing UV lesions in DNA and its absence results in the variant form of the genetic disorder, xeroderma pigmentosum. Other TLS polymerases have specificities for different types of damage, but their precise roles inside the cell have not yet been established. These polymerases are located in replication factories during DNA replication and the polymerase sliding clamp PCNA plays an important role in mediating switching between different polymerases.     

DNA polymerase mutagenic bypass and proofreading of endogenous DNA lesions

DNA polymerases differentiate between correct and incorrect substrates during synthesis on undamaged DNA templates through the biochemical steps of base incorporation, primer-template extension and proofreading excision. Recent research examining DNA polymerase processing of abasic, alkylation and oxidative lesions is reviewed in light of these discrimination mechanisms. Inhibition of DNA synthesis results from correct polymerase discrimination against utilization of geometrically incorrect template bases or 3' terminal basepairs. The efficiency of translesion synthesis is thus related to the physical structure of the lesion containing DNA. However, variations in enzyme structure and kinetics result in translesion synthesis efficiencies that are also dependent upon the DNA polymerase. With a low probability, polymerase misinsertion events create a 3' lesion terminus which is geometrically favored over the correct lesion basepair, resulting in mutagenic translesion synthesis. For example, both polymerase alpha and polymerase beta appear to require the formation of a stable 3' primer-template structure for efficient abasic site translesion synthesis. However, the enzymes differ as to the precise molecular make-up of the stable DNA structure, resulting in different mutational specificities. Similar mechanisms may be applicable to oxidative damage, where mutational specificities dependent upon the DNA polymerase also have been observed. In vitro reaction conditions also influence DNA polymerase processing of lesions. Using an in vitro herpes simplex virus thymidine kinase (HSV-tk) gene forward mutation assay, we demonstrate that high dNTP substrate concentrations affect the mutagenic specificity of translesion synthesis using alkylated templates. The exonuclease-deficient Klenow polymerase error frequency for G-->A transition mutations using templates modified by N-ethyl-N-nitrosourea (ENU) was four-fold higher at 1000 microM [dNTP], relative to 50 microM [dNTP], consistent with an increased efficiency of extension of the etO6G.T mispair. Moreover, the frequency of other ENU-induced polymerase errors was suppressed when polymerase reactions contained 50 microM dNTP, relative to 1000 microM dNTP. The efficiency of proofreading as a polymerase error discrimination mechanism reflects a balance between the competing processes of 3'-->5' exonuclease removal of mispairs and polymerization of the next correct nucleotide. Polymerases that are devoid of a proofreading exonuclease generally display enhanced abasic site translesion synthesis relative to proofreading-proficient enzymes. In addition, the proofreading exonucleases of Escherichia coli Pol I and T4 DNA polymerases have been found to remove mispairs caused by abasic sites and oxidative lesions, respectively, resulting in lowered polymerase error rates. However, the magnitude of the exonuclease effect is small (less than 10-fold), and highly dependent upon the DNA polymerase-exonuclease. We have studied proofreading exonuclease removal of alkylation damage in the HSV-tk forward assay. We observed no significant reduction in the magnitude of the mutant frequency vs. dose-response curves when N-methyl-N-nitrosourea or ENU-treated templates were used in exonuclease-proficient Klenow polymerase reactions, as compared to the exonuclease-deficient polymerase reactions. Thus, available data suggest that proofreading excision of endogenous lesion mispairs does occur, but the efficiency is dependent upon the lesion and the DNA polymerase-exonuclease studied.     

Evidence that DNA polymerases alpha and beta participate differentially in DNA repair synthesis induced by different agents

The roles of DNA polymerases alpha and beta in DNA replication and repair synthesis were studied in permeable animal cells, using different agents to induce repair synthesis. DNA polymerase inhibitors were used to investigate which polymerases were involved in repair synthesis and in replication. Polymerase alpha was responsible for replication. On the other hand, both polymerases alpha and beta were involved in DNA repair synthesis; the extent to which each polymerase participated depended primarily on the agent used to damage DNA. Polymerase beta was primarily responsible for repair synthesis induced by bleomycin or neocarzinostatin, whereas polymerase alpha played a more prominent role in repair synthesis indiced by N-methyl-N'-nitro-N-nitrosoguanidine or N-nitrosomethyl urea. More DNA damage was induced by the alkylating agents than by bleomycin or neocarzinostatin, suggesting that the extent of involvement of polymerase alpha or beta in DNA repair synthesis is related to the amount or type of DNA damage. In addition, salt concentration was found to have little or no effect on the results obtained with the DNA polymerase inhibitors. Our findings provide an explanation for conflicting reports in the literature concerning the roles of DNA polymerases alpha and beta in DNA repair.     

Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, alpha and delta.

DNA synthesis by two eukaryotic DNA polymerases, alpha and delta, was studied using a single-strand M13 DNA template primed at a unique site. In the presence of low amounts of either DNA polymerase alpha or delta, DNA synthesis was limited and short DNA strands of approximately 100 bases were produced. Addition of replication factors RF-A, PCNA and RF-C, which were previously shown to be required for SV40 DNA replication in vitro, differentially stimulated the activity of both DNA polymerases. RF-A and RF-C independently stimulated DNA polymerase alpha activity 4- to 6-fold, yielding relatively short DNA strands (less than 1 kb) and PCNA had no effect. In contrast, polymerase delta activity was stimulated co-operatively by PCNA, RF-A and RF-C approximately 25- to 30-fold, yielding relatively long DNA strands (up to 4 kb). Neither RF-C nor RF-A appear to correspond to known polymerase stimulatory factors. RF-A was previously shown to be required for initiation of DNA replication at the SV40 origin. Results presented here suggest that it also functions during elongation. The differential effects of these three replication factors on DNA polymerases alpha and delta is consistent with the model that the polymerases function at the replication fork on the lagging and leading strand templates respectively. We further suggest that co-ordinated synthesis of these strands requires dynamic protein-protein interactions between these replication factors and the two DNA polymerases.     

Distinct function of conserved amino acids in the fingers of Saccharomyces cerevisiae DNA polymerase alpha

Structural differences between class A and B DNA polymerases suggest that the motif B region, a wall of the catalytic pocket, may have evolved differentially in the two polymerase families. This study examines the function of the motif B residues in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha). Effects of the mutations were determined by biochemical analysis and genetic complementation of a yeast strain carrying a temperature-sensitive pol alpha mutant. Many conserved residues were viable with a variety of substitutions. Among them, mutations at Asn-948 or Tyr-951 conferred up to 8-fold higher colony formation frequency in a URA3 forward mutation assay, and 79-fold higher trp1 reversion frequency was observed for Y951P in yeast. Purified Y951P was as accurate as wild type in DNA synthesis but approximately 6-fold less processive and 22-fold less active in vitro. Therefore, Y951P may increase the frequency of mutant colony formation because of its low level of DNA polymerase activity in yeast. Mutations at Lys-944 or Gly-952 were not viable, which is consistent with the observation that mutants with substitutions at Gly-952 have strongly reduced catalytic activity in vitro. Gly-952 may provide a space for the nascent base pair and thus may play an essential function in S. cerevisiae DNA pol alpha. These results suggest that class B DNA polymerases have a unique structure in the catalytic pocket, which is distinct from the corresponding region in class A DNA polymerases.     

Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase eta (poleta), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase iota (poliota), like poleta, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that poleta and poliota foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poliota in replication foci is largely dependent on the presence of poleta. Using several different approaches, we demonstrate that poleta and poliota interact with each other physically and that the C-terminal 224 amino acids of poliota are sufficient for both the interaction with poleta and accumulation in replication foci. Our results provide strong evidence that poleta targets poliota to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.     

Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase eta (poleta), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase iota (poliota), like poleta, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that poleta and poliota foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poliota in replication foci is largely dependent on the presence of poleta. Using several different approaches, we demonstrate that poleta and poliota interact with each other physically and that the C-terminal 224 amino acids of poliota are sufficient for both the interaction with poleta and accumulation in replication foci. Our results provide strong evidence that poleta targets poliota to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.     

Mutagenic and recombinagenic responses to defective DNA polymerase delta are facilitated by the Rev1 protein in pol3-t mutants of Saccharomyces cerevisiae

Defective DNA replication can result in substantial increases in the level of genome instability. In the yeast Saccharomyces cerevisiae, the pol3-t allele confers a defect in the catalytic subunit of replicative DNA polymerase delta that results in increased rates of mutagenesis, recombination, and chromosome loss, perhaps by increasing the rate of replicative polymerase failure. The translesion polymerases Pol eta, Pol zeta, and Rev1 are part of a suite of factors in yeast that can act at sites of replicative polymerase failure. While mutants defective in the translesion polymerases alone displayed few defects, loss of Rev1 was found to suppress the increased rates of spontaneous mutation, recombination, and chromosome loss observed in pol3-t mutants. These results suggest that Rev1 may be involved in facilitating mutagenic and recombinagenic responses to the failure of Pol delta. Genome stability, therefore, may reflect a dynamic relationship between primary and auxiliary DNA polymerases.     

DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.     

The mutagenesis protein UmuC is a DNA polymerase activated by UmuD', RecA, and SSB and is specialized for translesion replication.

Replication of DNA lesions leads to the formation of mutations. In Escherichia coli this process is regulated by the SOS stress response, and requires the mutagenesis proteins UmuC and UmuD'. Analysis of translesion replication using a recently reconstituted in vitro system (Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199) revealed that lesion bypass occurred with a UmuC fusion protein, UmuD', RecA, and SSB in the absence of added DNA polymerase. Further analysis revealed that UmuC was a DNA polymerase (E. coli DNA polymerase V), with a weak polymerizing activity. Upon addition of UmuD', RecA, and SSB, the UmuC DNA polymerase was greatly activated, and replicated a synthetic abasic site with great efficiency (45% bypass in 6 min), 10-100-fold higher than E. coli DNA polymerases I, II, or III holoenzyme. Analysis of bypass products revealed insertion of primarily dAMP (69%), and to a lesser degree dGMP (31%) opposite the abasic site. The UmuC104 mutant protein was defective both in lesion bypass and in DNA synthesis. These results indicate that UmuC is a UmuD'-, RecA-, and SSB-activated DNA polymerase, which is specialized for lesion bypass. UmuC is a member of a new family of DNA polymerases which are specialized for lesion bypass, and include the yeast RAD30 and the human XP-V genes, encoding DNA polymerase eta.     

Sloppier copier DNA polymerases involved in genome repair

When chromosomal replication is impeded in the presence of DNA damage, members of a newly discovered UmuC/DinB/Rev1/Rad30 superfamily of procaryotic and eucaryotic DNA polymerases catalyze translesion synthesis at blocked replication forks. Although these polymerases share sequence elements essentially unrelated to the standard replication and repair enzymes, some of them (such as the SOS-induced Escherichia coli pol V) catalyze 'error-prone' translesion synthesis leading to large increases in mutation, whereas others (an example being the Xeroderma pigmentosum variant gene product XPV pol eta) carry out aberrant, yet nonmutagenic translesion synthesis. Ongoing studies of these low fidelity polymerases could provide new insights into the mechanism of somatic hypermutation, a key element in the immune response.