Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity.

Xeroderma pigmentosum variant (XP-V) represents one of the most common forms of this cancer-prone DNA repair syndrome. Unlike classical XP cells, XP-V cells are normal in nucleotide excision repair but defective in post-replication repair. The precise molecular defect in XP-V is currently unknown, but it appears to be a protein involved in translesion synthesis. Here we established a sensitive assay system using an SV40 origin-based plasmid to detect XP-V complementation activity. Using this system, we isolated a protein from HeLa cells capable of complementing the defects in XP-V cell extracts. The protein displays novel DNA polymerase activity which replicates cyclobutane pyrimidine dimer-containing DNA templates. The XPV polymerase activity was dependent on MgCl2, sensitive to NEM, moderately sensitive to KCl, resistant to both aphidicolin and ddTTP, and not stimulated by PCNA. In glycerol density gradients, the activity co-sedimented with a 54 kDa polypeptide at 3.5S, indicating that the monomeric form of this polypeptide was responsible for the activity. The protein factor corrected the translesion defects of extracts from three XPV cell strains. Bypass DNA synthesis by the XP-V polymerase occurred only in the presence of dATP, indicating that it can incorporate only dATP to bypass a di-thymine lesion.     

Adenovirus mediated transduction of the human DNA polymerase eta cDNA

Xeroderma pigmentosum (XP) is an autosomal recessive photosensitive disorder with an extremely high incidence of skin cancers. Seven complementation groups, corresponding to seven proteins involved in nucleotide excision repair (NER), are associated with this syndrome. However, in XP variant patients, the disorder is caused by defects in DNA polymerase eta; this error prone polymerase, encoded by POLH, is involved in translesion DNA synthesis (TLS) on DNA templates damaged by ultraviolet light (UV). We constructed a recombinant adenovirus carrying the human POLH cDNA linked to the EGFP reporter gene (AdXPV-EGFP) and infected skin fibroblasts from both XPV and XPA patients. Twenty-four hours after infection, the DNA polymerase eta-EGFP fusion protein was detected by Western blot analysis, demonstrating successful transduction by the adenoviral vector. Protein expression was accompanied by reduction in the high sensitivity of XPV cells to UV, as determined by cell survival and apoptosis-induction assays. Moreover, the pronounced UV-induced inhibition of DNA synthesis in XPV cells and their arrest in S phase were attenuated in AdXPV-EGFP infected cells, confirming that the transduced polymerase was functional. However, over-expression of polymerase eta mediated by AdXPV-EGFP infection did not result in enhancement of cell survival, prevention of apoptosis, or higher rate of nascent DNA strand growth in irradiated XPA cells. These results suggest that TLS by DNA polymerase eta is not a limiting factor for recovery from cellular responses induced by UV in excision-repair deficient fibroblasts.     

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.     

Asymmetry of DNA replication and translesion synthesis of UV-induced thymine dimers

In vitro replication assays for detection and quantification of bypass of UV-induced DNA photoproducts were used to compare the capacity of extracts prepared from different human cell lines to replicate past the cis,syn cyclobutane thymine dimer ([c,s]TT). The results demonstrated that neither nucleotide excision repair (NER) nor mismatch repair (MMR) activities in the intact cells interfered with measurements of bypass replication efficiencies in vitro. Extracts prepared from HeLa (NER- and MMR-proficient), xeroderma pigmentosum group A (NER-deficient), and HCT116 (MMR-deficient) cells displayed similar capacity for translesion synthesis, when the substrate carried the site-specific [c,s]TT on the template for the leading or the lagging strand of nascent DNA. Extracts from xeroderma pigmentosum variant cells, which lack DNA polymerase eta, were devoid of bypass activity. Bypass-proficient extracts as a group (n=16 for 3 extracts) displayed higher efficiency (P=0.005) for replication past the [c,s]TT during leading strand synthesis (84+/-22%) than during lagging strand synthesis (64+/-13%). These findings are compared to previous results concerning the bypass of the (6-4) photoproduct [Biochemistry 40 (2001) 15215] and analyzed in the context of the reported characteristics of bypass DNA polymerases implicated in translesion synthesis of UV-induced DNA lesions. Models to explain how these enzymes might interact with the DNA replication machinery are considered. An alternative pathway of bypass replication, which avoids translesion synthesis, and the mutagenic potential of post-replication repair mechanisms that contribute to the duplication of the human genome damaged by UV are discussed.     

Impaired translesion synthesis in xeroderma pigmentosum variant extracts

Xeroderma pigmentosum variant (XPV) cells are characterized by a cellular defect in the ability to synthesize intact daughter DNA strands on damaged templates. Molecular mechanisms that facilitate replication fork progression on damaged DNA in normal cells are not well defined. In this study, we used single-stranded plasmid molecules containing a single N-2-acetylaminofluorene (AAF) adduct to analyze translesion synthesis (TLS) catalyzed by extracts of either normal or XPV primary skin fibroblasts. In one of the substrates, the single AAF adduct was located at the 3' end of a run of three guanines that was previously shown to induce deletion of one G by a slippage mechanism. Primer extension reactions performed by normal cellular extracts from four different individuals produced the same distinct pattern of TLS, with over 80% of the products resulting from the elongation of a slipped intermediate and the remaining 20% resulting from a nonslipped intermediate. In contrast, with cellular extracts from five different XPV patients, the TLS reaction was strongly reduced, yielding only low amounts of TLS via the nonslipped intermediate. With our second substrate, in which the AAF adduct was located at the first G in the run, thus preventing slippage from occurring, we confirmed that normal extracts were able to perform TLS 10-fold more efficiently than XPV extracts. These data demonstrate unequivocally that the defect in XPV cells resides in translesion synthesis independently of the slippage process.     

Fidelity of human DNA polymerase eta

Xeroderma pigmentosum (XP) patients are highly sensitive to sunlight, and they suffer from a high incidence of skin cancers. The variant form of XP results from mutations in the hRAD30A gene, which encodes the DNA polymerase in humans, hPol(eta). Of the eukaryotic DNA polymerases, only human Pol(eta) and its yeast counterpart have the ability to replicate DNA containing a cis-syn thymine-thymine (T-T) dimer. Here we measure the fidelity of hPol(eta) on all four nondamaged template bases and at each thymine residue of a cis-syn T-T dimer. Opposite all four nondamaged template bases, hPol(eta) misincorporates nucleotides with a frequency of approximately 10(-2)-10(-3), and importantly, hPol(eta) synthesizes DNA opposite the T-T dimer with the same accuracy and efficiency as opposite the nondamaged DNA. The low fidelity of hPol(eta) may derive from a flexible active site that renders the enzyme more tolerant of geometric distortions in DNA and enables it to synthesize DNA past a T-T dimer.     

Pirh2 E3 ubiquitin ligase monoubiquitinates DNA polymerase eta to suppress translesion DNA synthesis

Polymerase eta (PolH) is necessary for translesion DNA synthesis, and PolH deficiency predisposes xeroderma pigmentosum variant (XPV) patients to cancer. Due to the critical role of PolH in translesion DNA synthesis, the activity of PolH is tightly controlled and subjected to multiple regulations, especially posttranslational modifications. Here, we show that PolH-dependent lesion bypass and intracellular translocation are regulated by Pirh2 E3 ubiquitin ligase through monoubiquitination. Specifically, we show that Pirh2, a target of the p53 tumor suppressor, monoubiquitinates PolH at one of multiple lysine residues. We also show that monoubiquitination of PolH inhibits the ability of PolH to interact with PCNA and to bypass UV-induced lesions, leading to decreased viability of UV-damaged cells. Moreover, we show that monoubiquitination of PolH alters the ability of PolH to translocate to replication foci for translesion DNA synthesis of UV-induced DNA lesions. Considering that Pirh2 is known to be overexpressed in various cancers, we postulate that in addition to mutation of PolH in XPV patients, inactivation of PolH by Pirh2 via monoubiquitination is one of the mechanisms by which PolH function is controlled, which might be responsible for the development and progression of some spontaneous tumors wherein PolH is not found to be mutated.     

The oxidative DNA lesion 8,5'-(S)-cyclo-2'-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.     

Translesion replication in cisplatin-treated xeroderma pigmentosum variant cells is also caffeine-sensitive: features of the error-prone DNA polymerase(s) involved in UV-mutagenesis

Patients with xeroderma pigmentosum variant (XP-V) have a higher risk to skin cancer and XP-V cells are extremely mutable by ultraviolet (UV). The defective gene encodes a DNA polymerase (Poleta) which catalyzed relatively accurate translesion synthesis past the cyclobutane dimer of UV-lesions instead of the replicative polymerase(s) that stalled just before the lesion. Pulse-chase studies have shown that translesion replication in XP-V cells is delayed, but does not completely cease. Taking these results together, error-prone polymerase(s) are plausively involved in the UV-mutagenesis in XP-V devoid of Poleta. However, less is known about the polymerase(s) in vivo. Using an alkaline sucrose density gradient centrifugation (ASDG) technique, translesion replication is detected in the two XP-V strains XP30RO and XP115LO. As reported by Lehmann et al. [Proc. Natl. Acad. Sci. U.S.A. 72 (1975): 219] in XP-V; (i) smaller replication products were accumulated after UV irradiation; (ii) the elongation of these products was delayed; (iii) the elongation was markedly inhibited by caffeine. XP-V cells UV-irradiated at mid-S phase were normally S-arrested, and no "override" by caffeine (i.e. abrogation of the S-checkpoint) was observed by flow cytometry, suggesting that caffeine does not act via cdc kinase here; (iv) butylphenyldeoxyguanosine (BuPGdR) inhibited elongation of replication products only in UV-irradiated XP-V cells; (v) dideoxycytidine or dideoxyinosine had no effect on this process in either normal or XP-V cells. Next, similar phenomena to UV (all of above i to v) were observed also in cisplatin-treated XP-V cells. Pol eta was indicated to participate in cisplatin-induced translesion replication in normal cells. Summing up the above results, the polymerase(s) which work in translesion replication in XP-V are probably BuPGdR-sensitive, insensitive to dideoxynucleotides and can bypass also cisplatin-lesions. To date, several polymerases capable of lesion-bypass synthesis have been isolated. The features presented here are quite useful for identifying the error-prone polymerase(s) involved in UV-mutagenesis.     

Reduced efficiency and increased mutagenicity of translesion DNA synthesis across a TT cyclobutane pyrimidine dimer, but not a TT 6-4 photoproduct, in human cells lacking DNA polymerase eta

Xeroderma pigmentosum variant (XPV) patients carry germ-line mutations in DNA polymerase eta (poleta), a major translesion DNA synthesis (TLS) polymerase, and exhibit severe sunlight sensitivity and high predisposition to skin cancer. Using a quantitative TLS assay system based on gapped plasmids we analyzed TLS across a site-specific TT CPD (thymine-thymine cyclobutane pyrimidine dimer) or TT 6-4 PP (thymine-thymine 6-4 photoproduct) in three pairs of poleta-proficient and deficient human cells. TLS across the TT CPD lesion was reduced by 2.6-4.4-fold in cells lacking poleta, and exhibited a strong 6-17-fold increase in mutation frequency at the TT CPD. All targeted mutations (74%) in poleta-deficient cells were opposite the 3'T of the CPD, however, a significant fraction (23%) were semi-targeted to the nearest nucleotides flanking the CPD. Deletions and insertions were observed at a low frequency, which increased in the absence of poleta, consistent with the formation of double strand breaks due to defective TLS. TLS across TT 6-4 PP was about twofold lower than across CPD, and was marginally reduced in poleta-deficient cells. TLS across TT 6-4 PP was highly mutagenic (27-63%), with multiple mutations types, and no significant difference between cells with or without poleta. Approximately 50% of the mutations formed were semi-targeted, of which 84-93% were due to the insertion of an A opposite the template G 5' to the 6-4 PP. These results, which are consistent with the UV hyper-mutability of XPV cells, highlight the critical role of poleta in error-free TLS across CPD in human cells, and suggest a potential involvement, although minor, of poleta in TLS across 6-4 PP under some conditions.     

Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences (<1 J/m², producing less than one pyrimidine dimer per replicon) rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates without noticeably affecting synthesis in multi-repliconsize intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher (>1 J/m², producing more than one dimer per replicon) cytotoxic fluences inhibited DNA synthesis in operating replicons presumably because the elongation of nascent strands was blocked where pyrimidine dimers were present in template strands. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences. indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation than the repair-deficient strains despite their ability to remove pyrimidine dimers. This analysis suggests that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery.     

Xeroderma pigmentosum complementation group E protein (XPE/DDB2): purification of various complexes of XPE and analyses of their damaged DNA binding and putative DNA repair properties

Xeroderma pigmentosum is characterized by increased sensitivity of the affected individuals to sunlight and light-induced skin cancers and, in some cases, to neurological abnormalities. The disease is caused by a mutation in genes XPA through XPG and the XP variant (XPV) gene. The proteins encoded by the XPA, -B, -C, -D, -F, and -G genes are required for nucleotide excision repair, and the XPV gene encodes DNA polymerase eta, which carries out translesion DNA synthesis. In contrast, the mechanism by which the XPE gene product prevents sunlight-induced cancers is not known. The gene (XPE/DDB2) encodes the small subunit of a heterodimeric DNA binding protein with high affinity to UV-damaged DNA (UV-damaged DNA binding protein [UV-DDB]). The DDB2 protein exists in at least four forms in the cell: monomeric DDB2, DDB1-DDB2 heterodimer (UV-DDB), and as a protein associated with both the Cullin 4A (CUL4A) complex and the COP9 signalosome. To better define the role of DDB2 in the cellular response to DNA damage, we purified all four forms of DDB2 and analyzed their DNA binding properties and their effects on mammalian nucleotide excision repair. We find that DDB2 has an intrinsic damaged DNA binding activity and that under our assay conditions neither DDB2 nor complexes that contain DDB2 (UV-DDB, CUL4A, and COP9) participate in nucleotide excision repair carried out by the six-factor human excision nuclease.     

Modeling xeroderma pigmentosum associated neurological pathologies with patients-derived iPSCs

Xeroderma pigmentosum (XP) is a group of genetic disorders caused by mutations of XP-associated genes, resulting in impairment of DNA repair. XP patients frequently exhibit neurological degeneration, but the underlying mechanism is unknown, in part due to lack of proper disease models. Here, we generated patientspecific induced pluripotent stem cells (iPSCs) harboring mutations in five different XP genes including XPA, XPB, XPC, XPG, and XPV. These iPSCs were further differentiated to neural cells, and their susceptibility to DNA damage stress was investigated. Mutation of XPA in either neural stem cells (NSCs) or neurons resulted in severe DNA damage repair defects, and these neural cells with mutant XPA were hyper-sensitive to DNA damage-induced apoptosis. Thus, XP-mutant neural cells represent valuable tools to clarify the molecular mechanisms of neurological abnormalities in the XP patients.     

Overproduction of DNA polymerase eta does not raise the spontaneous mutation rate in diploid human fibroblasts

Telomerase-immortalized lines of diploid xeroderma pigmentosum variant (XP-V) fibroblasts (XP115LO and XP4BE) were complemented for constitutive or regulated expression of wild-type human DNA polymerase eta (hpol eta). The ectopic gene was expressed from a retroviral LTR at a population average of 34- to 59-fold above the endogenous (mutated) mRNA and high levels of hpol eta were detected by immunoblotting. The POLH cDNA was also cloned downstream from an ecdysone-regulated promoter and transduced into the same recipient cells. Abundance of the wild-type mRNA increased approximately 10-fold by addition of ponasterone to the culture medium. Complemented cell lines acquired normal resistance to the cytotoxic effects of UVC, even in the presence of 1mM caffeine. They also tolerated higher levels of UVC-induced template lesions during nascent DNA elongation when compared to normal fibroblasts (NHF). UVC-induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus were measured in the XP115LO+XPV cell line overproducing hpol eta constitutively (E. Bassett, N.M. King, M.F. Bryant, S. Hector, L. Pendyala, S.G. Chaney, M. Cordeiro-Stone, The role of DNA polymerase eta in translesion synthesis past platinum-DNA adducts in human fibroblasts, Cancer Res. 64 (2004) 6469-6475). Induced mutation frequencies were significantly reduced, even below those observed in NHF; however, the average mutation frequency in untreated cultures was about three-fold higher than in the isogenic vector-control cell line. In this study, spontaneous HPRT mutation frequencies were measured at regular intervals, as isogenic fibroblasts either lacking or overproducing hpol eta were expanded for 100 population doublings. The mutation rates estimated from these results were not significantly increased in XP115LO cells expressing abnormal levels of hpol eta, relative to the cells lacking this specialized polymerase. These findings suggest that diploid human fibroblasts with normal DNA repair capacities and intact checkpoints are well protected against the potential mutagenic outcome of overproducing hpol eta, while still benefiting from accurate translesion synthesis of UV-induced pyrimidine dimers.     

Abnormal, Error-Prone Bypass of Photoproducts by Xeroderma Pigmentosum Variant Cell Extracts Results in Extreme Strand Bias for the Kinds of Mutations Induced by UV Light

Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a greatly increased susceptibility to sunlight-induced skin cancer. Cells from the majority of patients are defective in nucleotide excision repair. However, cells from one set of patients, XP variants, exhibit normal repair but are abnormally slow in replicating DNA containing UV photoproducts. The frequency of UV radiation-induced mutations in the XP variant cells is significantly higher than that in normal human cells. Furthermore, the kinds of UV-induced mutations differ very significantly from normal. Instead of transitions, mainly C→T, 30% of the base substitutions consist of C→A transversions, all arising from photoproducts located in one strand. Mutations involving cytosine in the other strand are almost all C→T transitions. Forty-five percent of the substitutions involve thymine, and the majority are transversions. To test the hypothesis that the UV hypermutability and the abnormal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells with those from HeLa cells and a fibroblast cell strain, MSU-1.2, for the ability to replicate a UV-irradiated form I M13 phage. The M13 template contains a simian virus 40 origin of replication located directly to the left or to the right of the target gene, lacZα, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to ~37% of the control value required only 1 photoproduct per template for XP variant cell extracts, but ~2.2 photoproducts for HeLa or MSU-1.2 cell extracts. The frequency of mutants induced was four times higher with XP variant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP variant cell extracts, the proportion of C→A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the template for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, this value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, and virtually all were T→A transversions. Sequence analysis of the coding region of the catalytic subunit of DNA polymerase delta in XP variant cell lines revealed two polymorphisms, but these do not account for the reduced bypass fidelity. Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cells, bypass of photoproducts involving cytosine in the template for the leading strand differs significantly from that of photoproducts in the lagging strand.     

Defective replication of psoralen adducts detected at the gene-specific level in xeroderma pigmentosum variant cells.

Replication of damaged DNA is suspected to play an important role in cell cycle, genetic stability, and survival pathways. Using psoralen photoaddition as prototype DNA damage and the renaturing agarose gel electrophoresis technique to measure DNA cross-linking in individual genes, Vos and Hanawalt previously observed efficient bypass replication of psoralen monoadducts in human genes (J.-M. H. Vos and P. C. Hanawalt, Cell 50:789-799, 1987). To understand the mechanism of bypass replication in human cells, mutants affected in such a process would be useful. We now report that cells from individuals suffering from the hereditary recessive syndrome xeroderma pigmentosum variant (XPV) are hypersensitive to killing induced by photoactivated psoralen. In addition, analysis of psoralen-mediated DNA cross-linking in the rRNA genes indicated that although repair of psoralen adducts was similar to that of normal individuals, XPV cells were markedly deficient in the ability to bypass psoralen adducts during replication; in comparison with normal cells, approximately half as many monoadducts were bypassed during replication in XPV cells. Furthermore, in contrast to normal cells, replication of interstrand cross-links was not detected in XPV. This is the first demonstration of a deficiency in bypass replication detected at the gene-specific level in vivo. A model involving a strand-specific defect in recombinational bypass in XPV is proposed.     

Polymerase eta and p53 jointly regulate cell survival,apoptosis and Mre11 recombination during S phase checkpoint arrest after UV irradiation

Xeroderma pigmentosum variant (XPV) cells lack the damage-specific polymerase eta and undergo a protracted arrest at the S phase checkpoint(s) following UV damage. The S phase checkpoints encompass several qualitatively different processes, and stimulate downstream events that are dependent on the functional state of p53. Primary fibroblasts with wild-type p53 arrest in S, and require a functional polymerase eta (pol eta) to carry out bypass replication, but do not recruit recombination factors for recovery. XPV cells with non-functional p53, as a result of transformation by SV40 or HPV16 (E6/E7), recruit the hMre11/hRad50/Nbs1 complex to arrested replication forks, coincident with PCNA, whereas normal transformed cells preferentially use the pol eta bypass replication pathway. The formation of hMre11 foci implies that arrested replication forks rapidly undergo a collapse involving double strand breakage and rejoining. Apoptosis occurs after UV only in cells transformed by SV40, and not in normal or XPV fibroblasts or HPV16 (E6/E7) transformed cells. Conversely, ultimate cell survival in XPV cells was much less in HPV16 (E6/E7) transformed cells than in SV40 transformed cells, indicating that apoptosis was not a reliable predictor of cell survival. Inhibition of p53 transactivation by pifithrin-alpha or inhibition of protein synthesis by cycloheximide did not induce hMre11 foci or apoptosis in UV damaged fibroblasts. Inhibition of kinase activity with wortmannin did not increase killing by UV, unlike the large increase seen with caffeine. Since HPV16 (E6/E7) transformed XPV cells were highly UV sensitive and not further sensitized by caffeine, it appears likely that caffeine sensitization proceeds through a p53 pathway. The S phase checkpoints are therefore, a complex set of different checkpoints that are coordinated by p53 with the capacity to differentially modulate cell survival, apoptosis, bypass replication and hMre11 recombination.     

Replicative bypass repair of ultraviolet damage to DNA of mammalian cells: Caffeine sensitive and caffeine resistant mechanisms

Replicative bypass repair of UV damage to DNA was studied in wide variety of human, mouse and hamster cells in culture. Survival curve analysis revealed that in established cell lines (mouse L, Chinese hamster V79, HeLa S3 and SV40-transformed xeroderma pigmentosum (XP)), post-UV caffeine treatment potentiated cell killing by reducing the extrapolation number and mean lethal UV fluence (Do). In the Do reduction as the result of random inactivation by caffeine of sensitive repair there were marked clonal differences among such cell lines, V79 being most sensitive to caffeine potentiation. However, other diploid cell lines (normal human, excision-defective XP and Syrian hamster) exhibited no obvious reduction in Do by caffeine. In parallel, alkaline sucrose sedimentation results showed that the conversion of initially smaller segments of DNA synthetized after irradiation with 10 J/m² to high-molecular-weight DNA was inhibited by caffeine in transformed XP cells, but not in the diploid human cell lines. Exceptionall, diploid XP variants had a retarded ability of bypass repair which was drastically prevented by caffeine, so that caffeine enhanced the lethal effect of UV. Neutral CsCl study on the bypass repair mechanism by use of bromodeoxyuridine for DNA synthesis on damaged template suggests that the pyrimidine dimer acts as a block to replication and subsequently it is circumvented presumably by a new process involving replicative bypassing following strand displacement, rather than by gap-filling de novo. This mechanism worked similarly in normal and XP cells, whether or not caffeine was present, indicating that excision of dimer is not always necessary. However, replicative became defective in XP variant and transformed XP cells when caffeine was present. It appears, therefore, that the replicative bypass repair process is either caffeine resistant or sensitive, depending on the cell type used, but not necessarily on the excision repair capability.     

Complementation of defective translesion synthesis and UV light sensitivity in xeroderma pigmentosum variant cells by human and mouse DNA polymerase eta

Defects in the human gene XPV result in the variant form of the genetic disease xeroderma pigmentosum (XP-V). XPV encodes DNA polymerase η, a novel DNA polymerase that belongs to the UmuC/DinB/Rad30 superfamily. This polymerase catalyzes the efficient and accurate translesion synthesis of DNA past cis-syn cyclobutane di-thymine lesions. In this report we present the cDNA sequence and expression profiles of the mouse XPV gene and demonstrate its ability to complement defective DNA synthesis in XP-V cells. The mouse XPV protein shares 80.3% amino acid identity and 86.9% similarity with the human XPV protein. The recombinant mouse XPV protein corrected the inability of XP-V cell extracts to carry out DNA replication, by bypassing thymine dimers on template DNA. Transfection of the mouse or human XPV cDNA into human XP-V cells corrected UV sensitivity. Northern blot analysis revealed that the mouse XPV gene is expressed ubiquitously, but at a higher level in testis, liver, skin and thymus compared to other tissues. Although the mouse XPV gene was not induced by UV irradiation, its expression was elevated ~4-fold during cell proliferation. These results suggest that DNA polymerase η plays a role in DNA replication, though the enzyme is not essential for viability.     

Mutagenic and nonmutagenic bypass of DNA lesions by Drosophila DNA polymerases dpoleta and dpoliota

cDNA sequences were identified and isolated that encode Drosophila homologues of human Rad30A and Rad30B called drad30A and drad30B. Here we show that the C-terminal-truncated forms of the drad30A and drad30B gene products, designated dpoletaDeltaC and dpoliotaDeltaC, respectively, exhibit DNA polymerase activity. dpoletaDeltaC and dpoliotaDeltaC efficiently bypass a cis-syn-cyclobutane thymine-thymine (TT) dimer in a mostly error-free manner. dpoletaDeltaC shows limited ability to bypass a 6-4-photoproduct ((6-4)PP) at thymine-thymine (TT-(6-4)PP) or at thymine-cytosine (TC-(6-4)PP) in an error-prone manner. dpoliotaDeltaC scarcely bypasses these lesions. Thus, the fidelity of translesion synthesis depends on the identity of the lesion and on the polymerase. The human XPV gene product, hpoleta, bypasses cis-syn-cyclobutane thymine-thymine dimer efficiently in a mostly error-free manner but does not bypass TT-(6-4)PP, whereas Escherichia coli DNA polymerase V (UmuD'(2)C complex) bypasses both lesions, especially TT-(6-4)PP, in an error-prone manner (Tang, M., Pham, P., Shen, X., Taylor, J. S., O'Donnell, M., Woodgate, R., and Goodman, M. F. (2000) Nature 404, 1014-1018). Both dpoletaDeltaC and DNA polymerase V preferentially incorporate GA opposite TT-(6-4)PP. The chemical structure of the lesions and the similarity in the nucleotides incorporated suggest that structural information in the altered bases contribute to nucleotide selection during incorporation opposite these lesions by these polymerases.