Xeroderma Pigmentosum Variant Patients Research Articles

Mutational signatures and increased retrotransposon insertions in xeroderma pigmentosum variant skin tumors.

Xeroderma pigmentosum variant (XP-V) is an autosomal recessive disease with an increased risk of developing cutaneous neoplasms in sunlight-exposed regions. These cells are deficient in the translesion synthesis (TLS) DNA polymerase eta, responsible for bypassing different types of DNA lesions. From the exome sequencing of 11 skin tumors of a genetic XP-V patients' cluster, classical mutational signatures related to sunlight exposure, such as C>T transitions targeted to pyrimidine dimers, were identified. However, basal cell carcinomas also showed distinct C>A mutation spectra reflecting a mutational signature possibly related to sunlight-induced oxidative stress. Moreover, four samples carry different mutational signatures, with C>A mutations associated with tobacco chewing or smoking usage. Thus, XP-V patients should be warned of the risk of these habits. Surprisingly, higher levels of retrotransposon somatic insertions were also detected when the tumors were compared with non-XP skin tumors, revealing other possible causes for XP-V tumors and novel functions for the TLS polymerase eta in suppressing retrotransposition. Finally, the expected high mutation burden found in most of these tumors renders these XP patients good candidates for checkpoint blockade immunotherapy.

The key role of UVA-light induced oxidative stress in human Xeroderma Pigmentosum Variant cells

The UVA component of sunlight induces DNA damage, which are basically responsible for skin cancer formation. Xeroderma Pigmentosum Variant (XP-V) patients are defective in the DNA polymerase pol eta that promotes translesion synthesis after sunlight-induced DNA damage, implying in a clinical phenotype of increased frequency of skin cancer. However, the role of UVA-light in the carcinogenesis of these patients is not completely understood. The goal of this work was to characterize UVA-induced DNA damage and the consequences to XP-V cells, compared to complemented cells. DNA damage were induced in both cells by UVA, but lesion removal was particularly affected in XP-V cells, possibly due to the oxidation of DNA repair proteins, as indicated by the increase of carbonylated proteins. Moreover, UVA irradiation promoted replication fork stalling and cell cycle arrest in the S-phase for XP-V cells. Interestingly, when cells were treated with the antioxidant N-acetylcysteine, all these deleterious effects were consistently reverted, revealing the role of oxidative stress in these processes. Together, these results strongly indicate the crucial role of oxidative stress in UVA-induced cytotoxicity and are of interest for the protection of XP-V patients.

Filling gaps in translesion DNA synthesis in human cells

During DNA replication, forks may encounter unrepaired lesions that hamper DNA synthesis. Cells have universal strategies to promote damage bypass allowing cells to survive. DNA damage tolerance can be performed upon template switch or by specialized DNA polymerases, known as translesion (TLS) polymerases. Human cells count on more than eleven TLS polymerases and this work reviews the functions of some of these enzymes: Rev1, Pol η, Pol ι, Pol κ, Pol θ and Pol ζ. The mechanisms of damage bypass vary according to the lesion, as well as to the TLS polymerases available, and may occur directly at the fork during replication. Alternatively, the lesion may be skipped, leaving a single-stranded DNA gap that will be replicated later. Details of the participation of these enzymes are revised for the replication of damaged template. TLS polymerases also have functions in other cellular processes. These include involvement in somatic hypermutation in immunoglobulin genes, direct participation in recombination and repair processes, and contributing to replicating noncanonical DNA structures. The importance of DNA damage replication to cell survival is supported by recent discoveries that certain genes encoding TLS polymerases are induced in response to DNA damaging agents, protecting cells from a subsequent challenge to DNA replication. We retrace the findings on these genotoxic (adaptive) responses of human cells and show the common aspects with the SOS responses in bacteria. Paradoxically, although TLS of DNA damage is normally an error prone mechanism, in general it protects from carcinogenesis, as evidenced by increased tumorigenesis in xeroderma pigmentosum variant patients, who are deficient in Pol η. As these TLS polymerases also promote cell survival, they constitute an important mechanism by which cancer cells acquire resistance to genotoxic chemotherapy. Therefore, the TLS polymerases are new potential targets for improving therapy against tumors.

129-Derived Mouse Strains Express an Unstable but Catalytically Active DNA Polymerase Iota Variant.

Mice derived from the 129 strain have a nonsense codon mutation in exon 2 of the polymerase iota (Polι) gene and are therefore considered Polι deficient. When we amplified Polι mRNA from 129/SvJ or 129/Ola testes, only a small fraction of the full-length cDNA contained the nonsense mutation; the major fraction corresponded to a variant Polι isoform lacking exon 2. Polι mRNA lacking exon 2 contains an open reading frame, and the corresponding protein was detected using a polyclonal antibody raised against the C terminus of the murine Polι protein. The identity of the corresponding protein was further confirmed by mass spectrometry. Although the variant protein was expressed at only 5 to 10% of the level of wild-type Polι, it retained de novo DNA synthesis activity, the capacity to form replication foci following UV irradiation, and the ability to rescue UV light sensitivity in Polι(-/-) embryonic fibroblasts derived from a new, fully deficient Polι knockout (KO) mouse line. Furthermore, in vivo treatment of 129-derived male mice with Velcade, a drug that inhibits proteasome function, stabilized and restored a substantial amount of the variant Polι in these animals, indicating that its turnover is controlled by the proteasome. An analysis of two xeroderma pigmentosum-variant (XPV) cases corresponding to missense mutants of Polη, a related translesion synthesis (TLS) polymerase in the same family, similarly showed a destabilization of the catalytically active mutant protein by the proteasome. Collectively, these data challenge the prevailing hypothesis that 129-derived strains of mice are completely deficient in Polι activity. The data also document, both for 129-derived mouse strains and for some XPV patients, new cases of genetic defects corresponding to the destabilization of an otherwise functional protein, the phenotype of which is reversible by proteasome inhibition.

Aberrant C-terminal domain of polymerase η targets the functional enzyme to the proteosomal degradation pathway.

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by sunlight sensitivity and predisposition to cutaneous malignancies. XP-V is caused by a deficiency in DNA polymerase eta (Polη) that plays a pivotal role in translesion synthesis by bypassing UV-induced pyrimidine dimers. Previously we identified a new Polη variant containing two missense mutations, one mutation within the bipartite NLS (T692A) and a second mutation on the stop codon (X714W) leading to a longer protein with an extra 8 amino acids (721 instead of 713 AA). First biochemical analysis revealed that this Polη missense variant was barely detectable by western blot. As this mutant is extremely unstable and is nearly undetectable, a definitive measure of its functional deficit in cells has not been explored. Here we report the molecular and cellular characterization of this missense variant. In cell free extracts, the extra 8 amino acids in the C-terminal of Polη(721) only slightly reduce the bypass efficiency through CPD lesions. In vivo, Polη(721) accumulates in replication factories and interacts with mUb-PCNA albeit at lower level than Polη(wt). XP-V cells overexpressing Polη(721) were only slightly UV-sensitive. Altogether, our data strongly suggest that Polη(721) is functional and that the patient displays a XP-V phenotype because the mutant protein is excessively unstable. We then investigated the molecular mechanisms involved in this excessive proteolysis. We showed that Polη(721) is degraded by the proteasome in an ubiquitin-dependent manner and that this proteolysis is independent of the E3 ligases, CRL4(cdt2) and Pirh2, reported to promote Polη degradation. We then demonstrated that the extra 8 amino acids of Polη(721) do not act as a degron but rather induce a conformational change of the Polη C-terminus exposing its bipartite NLS as well as a sequence close to its UBZ to the ubiquitin/proteasome system. Interestingly we showed that the clinically approved proteasome inhibitor, Bortezomib restores the levels of Polη(721) suggesting that this might be a therapeutic approach to preventing tumor development in certain XP-V patients harboring missense mutations.

Correlation of Phenotype/Genotype in a Cohort of 23 Xeroderma Pigmentosum-Variant Patients Reveals 12 New Disease-CausingPOLHMutations

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by some sunlight sensitivity and predisposition to cutaneous malignancies. We described clinical and genetic features of the largest collection ever published of 23 XPV patients (ages between 21 and 86) from 20 unrelated families. Primary fibroblasts from patients showed normal nucleotide excision repair but UV-hypersensitivity in the presence of caffeine, a signature of the XP-V syndrome. 87% of patients developed skin tumors with a median age of 21 for the first occurrence. The median numbers of basal-cell carcinoma was 13 per patient, six for squamous-cell carcinoma, and five for melanoma. XP-V is due to defects in the translesion-synthesis DNA polymerase Polη coded by the POLH gene. DNA sequencing of POLH revealed 29 mutations, where 12 have not been previously identified, leading to truncated polymerases in 69% of patients. Four missense mutations are correlated with the protein stability by structural modeling of the Polη polymerase domain. There is a clear relationship between the types of missense mutations and clinical severity. For truncating mutations, which lead to an absence of or to inactive proteins, the life-cumulated UV exposure is probably the best predictor of cancer incidence, reinforcing the necessity to protect XP-Vs from sun exposure.

A non-catalytic role of DNA polymerase η in recruiting Rad18 and promoting PCNA monoubiquitination at stalled replication forks

Trans-lesion DNA synthesis (TLS) is a DNA damage-tolerance mechanism that uses low-fidelity DNA polymerases to replicate damaged DNA. The inherited cancer-propensity syndrome xeroderma pigmentosum variant (XPV) results from error-prone TLS of UV-damaged DNA. TLS is initiated when the Rad6/Rad18 complex monoubiquitinates proliferating cell nuclear antigen (PCNA), but the basis for recruitment of Rad18 to PCNA is not completely understood. Here, we show that Rad18 is targeted to PCNA by DNA polymerase eta (Polη), the XPV gene product that is mutated in XPV patients. The C-terminal domain of Polη binds to both Rad18 and PCNA and promotes PCNA monoubiquitination, a function unique to Polη among Y-family TLS polymerases and dissociable from its catalytic activity. Importantly, XPV cells expressing full-length catalytically-inactive Polη exhibit increased recruitment of other error-prone TLS polymerases (Polκ and Polι) after UV irradiation. These results define a novel non-catalytic role for Polη in promoting PCNA monoubiquitination and provide a new potential mechanism for mutagenesis and genome instability in XPV individuals.

Identification of a Novel Nonsense Mutation in POLH in a Chinese Pedigree with Xeroderma Pigmentosum, Variant Type

Xeroderma pigmentosum-variant (XPV) is one type of XP, a rare autosomal recessive disorder, and caused by defects in the post replication repair machinery while nucleotide-excision repair (NER) is not impaired. In the present study, we reported a Chinese family with XPV phenotype, which was confirmed by histopathological results. Genetic variants were detected by polymerase chain reaction and exon sequencing. Furthermore, the reported molecular defects in XPV patients from previous literatures were reviewed. A homozygous c.67C>T mutation in the exon 2 of DNA polymerase eta (POLH), a novel non-sense mutation in POLH, was discovered.

Abstract 3813: DNA polymerase eta and a novel anticancer selenophene compound

Abstract Human DNA polymerase ≤ (pol α) plays a key role in replicating across the UV radiation induced cyclobutane pyrimidine dimers (CPD) in DNA and mutations in pol ≤ gene result in a syndrome called Xeroderma Pigmentosum Variant (XP-V). In the absence of pol β, the DNA replication forks collapse after prolonged stalling at CPD sites, which consequently lead to DNA breakages and cell death. Therefore, pol ≤ has been considered to serve as a cellular protection mechanism to enhance cell survival and reduce mutagenesis. In addition to UV-induced CPD lesion, pol ≤ has the ability to handle DNA lesions introduced by chemotherapeutic anticancer agents, such as platinum based compounds and nucleoside analogs. Such damage tolerance ability of pol ≤ has been shown to reduce cell sensitivity to these DNA targeting agents. D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-N-methylpyrrole] is a selenophene compounds that demonstrated promising antitumor activity against various human cancer cells and in vivo xenograft mouse model. Mechanistic studies have shown that D-501036 induced DNA adduct(s) and caused genome instability, which result in cell cycle accumulation in the S-phase, DNA breakage, and subsequently lead to cell death. Interestingly, the IC50 for human fibroblast XP30RO cells derived from XP-V patients was >50 fold higher than the XP30RO cells that stably transfected with a pol ≤ expressing vector (XP30RO-EGFP-pol α). In addition, D-501036 caused an increased amount of DNA breakage in the XP30RO-EGFP-pol ≤ cells as compared to its parental XP30RO cells in both DNA fragmentation and comet assays. These results revealed a novel role of pol ≤ in the action of D-501036 that is very different from its protective role against UV irradiation or cisplatin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3813. doi:1538-7445.AM2012-3813

Stalled Polη at its cognate substrate initiates an alternative translesion synthesis pathway via interaction with REV1

DNA polymerase η (Polη), whose gene mutation is responsible for the inherited disorder xeroderma pigmentosum variant (XP-V), carries out accurate and efficient translesion synthesis (TLS) across cyclobutane pyrimidine dimer (CPD). As Polη interacts with REV1, and REV1 interacts with other TLS polymerases including Polι, Polκ and Polζ, Polη may play a role in recruitment of these TLS polymerases at lesion site. But it is unclear whether UV sensitivity of XP-V patients is caused not only by defect of Polη activity but also by dysfunction of network between Polη and other TLS polymerases. Here, we examined whether the TLS polymerase network via Polη is important for replicative bypass of CPDs and DNA damage tolerance induced by UV in mouse cells. We observed that UV sensitivity of Polη-deficient mouse cells was moderately rescued by the expression of a catalytically inactive Polη. Moreover, this recovery of cellular UV sensitivity was mediated by the interaction between Polη and REV1. However, expression of the inactive mutant Polη was not able to suppress the incidence of UV-induced mutation observed in Polη-deficient cells. We propose the model that REV1 and Polκ are involved in DNA damage tolerance via Polη-REV1 interaction when Polη fails to bypass its cognate substrates.

DNA polymerase eta is targeted by Mdm2 for polyubiquitination and proteasomal degradation in response to ultraviolet irradiation

DNA polymerase eta (PolH), the product of the xeroderma pigmentosum variant (XPV) gene and a Y-family DNA polymerase, plays a pivotal role in translesion DNA synthesis. Loss of PolH leads to early onset of malignant skin cancer in XPV patients and increases UV-induced carcinogenesis. Thus, the pathways by which PolH expression and activity are controlled may be explored as a strategy to prevent UV-induced cancer. In this study, we found that Mdm2, a RING finger E3 ligase, promotes PolH degradation. Specifically, we showed that knockdown of Mdm2 increases PolH expression in both p53-proficient and -deficient cells. In addition, we showed that UV-induced PolH degradation is attenuated by Mdm2 knockdown. In contrast, ectopically expression of Mdm2 decreases PolH expression, which can be abrogated by the proteasome inhibitor MG132. Moreover, we showed that Mdm2 physically associates with PolH and promotes PolH polyubiquitination in vivo and in vitro. Finally, we showed that knockdown of Mdm2 increases the formation of PolH replication foci and decreases the sensitivity of cells to UV-induced lesions in a PolH-dependent manner. Taken together, we uncovered that Mdm2 serves as an E3 ligase for PolH polyubiquitination and proteasomal degradation in cells under the basal condition and in response to UV irradiation.

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.

Requirement for functional DNA polymerase eta in genome-wide repair of UV-induced DNA damage during S phase

The autosomal recessive disorder Xeroderma pigmentosum- variant (XPV) is characterized (i) at the cellular level by dramatic hypermutability and defective recovery of DNA synthesis following UV exposure, and (ii) clinically by abnormal sunlight sensitivity and remarkable predisposition to skin cancer. These phenotypes are clearly attributable to germline mutations in POLH, encoding DNA polymerase eta (polη) normally required for accurate translesion DNA synthesis (TLS) past UV-induced cyclobutane pyrimidine dimers. Here we demonstrate that patient-derived XPV-skin fibroblasts exposed to 15 J/m 2 of UV also exhibit (in addition to abnormal TLS) a significant defect in global-genomic nucleotide excision repair (GG-NER) exclusively during S phase. This cell cycle-specific GG-NER defect can be complemented by ectopic expression of wild-type polη, but not of polη variants deficient in either nuclear relocalization or PCNA interaction. We highlight a previous study from our laboratory demonstrating that UV-exposed, ATR-deficient Seckel syndrome fibroblasts, like XPV fibroblasts, manifest strong attenuation of GG-NER uniquely in S phase populations. We now present further evidence suggesting that deficient S phase repair can be rescued in both XPV- and Seckel syndrome-cells if the formation of blocked replication forks post-UV is either prevented or substantially reduced, i.e., following, respectively, pharmacological inhibition of DNA synthesis prior to UV irradiation, or exposure to a relatively low UV dose (5 J/m 2). Our findings in cultured cells permit speculation that abrogation of GG-NER during S phase might partially contribute (in a synergistic manner with defective, atypically error-prone TLS) to the extreme state of UV-hypermutability leading to accelerated skin cancer development in XPV patients. Moreover, based on the overall data, we postulate that loss of either functional polη or -ATR engenders abnormal persistence of stalled replication forks at UV-adducted sites in DNA which, in turn, can actively and/or passively trigger GG-NER inhibition.

DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase eta, the POLH gene product. A deficiency in DNA polymerase eta due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase zeta cooperates with DNA polymerases kappa and iota to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases zeta and kappa, but not iota, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.

Human DNA polymerase η activity and translocation is regulated by phosphorylation

Human DNA polymerase eta (pol eta) can replicate across UV-induced pyrimidine dimers, and defects in the gene encoding pol eta result in a syndrome called xeroderma pigmentosum variant (XP-V). XP-V patients are prone to the development of cancer in sun-exposed areas, and cells derived from XP-V patients demonstrate increased sensitivity to UV radiation and a higher mutation rate compared with wild-type cells. pol eta has been shown to replicate across a wide spectrum of DNA lesions introduced by environmental or chemotherapeutic agents, or during nucleotide starvation, suggesting that the biological roles for pol eta are not limited to repair of UV-damaged DNA. The high error rate of pol eta requires that its intracellular activity be tightly regulated. Here, we show that the phosphorylation of pol eta increased after UV irradiation, and that treatment with caffeine, siRNA against ATR, or an inhibitor of PKC (calphostin C), reduced the accumulation of pol eta at stalled replication forks after UV irradiation or treatment with cisplatin and gemcitabine. Site-specific mutagenesis (S587A and T617A) of pol eta at two putative PKC phosphorylation sites located in the protein-protein interaction domain prevented nuclear foci formation induced by UV irradiation or treatment with gemcitabine/cisplatin. In addition, XP-V cell lines stably expressing either the S587A or T617A mutant form of pol eta were more sensitive to UV radiation and gemcitabine/cisplatin than control cells expressing wild-type pol eta. These results suggest that phosphorylation is one mechanism by which the cellular activity of pol eta is regulated.

Xeroderma Pigmentosum-Variant Patients from America, Europe, and Asia

Xeroderma pigmentosum-variant (XP-V) patients have sun sensitivity and increased skin cancer risk. Their cells have normal nucleotide excision repair, but have defects in the POLH gene encoding an error-prone polymerase, DNA polymerase eta (pol eta). To survey the molecular basis of XP-V worldwide, we measured pol eta protein in skin fibroblasts from putative XP-V patients (aged 8-66 years) from 10 families in North America, Turkey, Israel, Germany, and Korea. Pol eta was undetectable in cells from patients in eight families, whereas two showed faint bands. DNA sequencing identified 10 different POLH mutations. There were two splicing, one nonsense, five frameshift (3 deletion and 2 insertion), and two missense mutations. Nine of these mutations involved the catalytic domain. Although affected siblings had similar clinical features, the relation between the clinical features and the mutations was not clear. POLH mRNA levels were normal or reduced by 50% in three cell strains with undetectable levels of pol eta protein, indicating that nonsense-mediated message decay was limited. We found a wide spectrum of mutations in the POLH gene among XP-V patients in different countries, suggesting that many of these mutations arose independently.

Mutation Frequencies and Spectra in DNA Polymerase η–Deficient Mice

The low-fidelity polymerase eta (poleta) is required for bypass of UV-induced pyrimidine dimers inserting adenine nucleotides opposite these lesions. Mutations in the poleta gene are responsible for the genetic defect in xeroderma pigmentosum variant patients. To study if the lack of poleta significantly elevates spontaneous mutation frequency in various organs and tissues of the mouse, we crossed poleta-deficient mice with transgenic mice harboring a chromosomally integrated lacZ-plasmid reporter construct. In cultured embryonic fibroblasts from the lacZ-poleta(-/-) mice, 2.5 J/m(2) UV irradiation induced approximately 5-fold more mutations than in cells from lacZ control mice, in which an approximately 3-fold increase in mutation frequency was found compared with the normal level. Whereas untreated cells harbored mainly 1-bp deletions, UV induced both transitions and transversions, with the latter type more highly represented in the poleta-null cells than in the controls. No difference in mutation induction between the poleta-null cells and the wild-type cells was observed after treatment with N-ethyl-N-nitrosourea. Having shown the validity of the lacZ model to accurately identify poleta-associated mutagenesis, we then determined the mutant frequency at the lacZ locus in liver, spleen, and small intestine of 12-month-old animals. No differences were found between poleta-null, heterozygous, or littermate control mice. We conclude that the poleta defect is specific for UV damage and has no effect on in vivo mutagenesis in mice.

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 η; 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 (Ad XPV- EGFP) and infected skin fibroblasts from both XPV and XPA patients. Twenty-four hours after infection, the DNA polymerase η-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 Ad XPV- EGFP infected cells, confirming that the transduced polymerase was functional. However, over-expression of polymerase η mediated by Ad XPV- 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 η is not a limiting factor for recovery from cellular responses induced by UV in excision-repair deficient fibroblasts.

DNA Polymerase η, the Product of the Xeroderma Pigmentosum Variant Gene and a Target of p53, Modulates the DNA Damage Checkpoint and p53 Activation

DNA polymerase eta (PolH) is the product of the xeroderma pigmentosum variant (XPV) gene and a well-characterized Y-family DNA polymerase for translesion synthesis. Cells derived from XPV patients are unable to faithfully bypass UV photoproducts and DNA adducts and thus acquire genetic mutations. Here, we found that PolH can be up-regulated by DNA breaks induced by ionizing radiation or chemotherapeutic agents, and knockdown of PolH gives cells resistance to apoptosis induced by DNA breaks in multiple cell lines and cell types in a p53-dependent manner. To explore the underlying mechanism, we examined p53 activation upon DNA breaks and found that p53 activation is impaired in PolH knockdown cells and PolH-null primary fibroblasts. Importantly, reconstitution of PolH into PolH knockdown cells restores p53 activation. Moreover, we provide evidence that, upon DNA breaks, PolH is partially colocalized with phosphorylated ATM at gamma-H2AX foci and knockdown of PolH impairs ATM to phosphorylate Chk2 and p53. However, upon DNA damage by UV, PolH knockdown cells exhibit two opposing temporal responses: at the early stage, knockdown of PolH suppresses p53 activation and gives cells resistance to UV-induced apoptosis in a p53-dependent manner; at the late stage, knockdown of PolH suppresses DNA repair, leading to sustained activation of p53 and increased susceptibility to apoptosis in both a p53-dependent and a p53-independent manner. Taken together, we found that PolH has a novel role in the DNA damage checkpoint and that a p53 target can modulate the DNA damage response and subsequently regulate p53 activation.

Contribution of DNA polymerase η to immunoglobulin gene hypermutation in the mouse

The mutation pattern of immunoglobulin genes was studied in mice deficient for DNA polymerase η, a translesional polymerase whose inactivation is responsible for the xeroderma pigmentosum variant (XP-V) syndrome in humans. Mutations show an 85% G/C biased pattern, similar to that reported for XP-V patients. Breeding these mice with animals harboring the stop codon mutation of the 129/Olain background in their DNA polymerase ι gene did not alter this pattern further. Although this G/C biased mutation profile resembles that of mice deficient in the MSH2 or MSH6 components of the mismatch repair complex, the residual A/T mutagenesis of polη-deficient mice differs markedly. This suggests that, in the absence of polη, the MSH2–MSH6 complex is able to recruit another DNA polymerase that is more accurate at copying A/T bases, possibly polκ, to assume its function in hypermutation.