Xeroderma Pigmentosum Variant Research Articles

Global Contributions to the Understanding of DNA Repair and Skin Cancer

Living organisms exposed to sunlight have developed mechanisms to repair the damage that UVR induces in their DNA. These DNA repair systems are present in most life forms including bacteria, yeast, rodents, and mammals. Three rare human genetic diseases have defects in the DNA repair system called nucleotide excision repair (NER). Patients with xeroderma pigmentosum (XP) have defective NER and a more than 10,000-fold increased frequency of skin cancer, whereas patients with Cockayne syndrome (CS) or with trichothiodystrophy (TTD) have NER defects without increase in cancer. These are strikingly different clinical phenotypes (Figure 1). The dramatic differences in their manifestations reflect the complexity of DNA repair and the broad scope and central role of DNA repair on preservation of diverse biological functions. This article will review the major milestones in our understanding of DNA repair and skin cancer (Table 1). These include recognition of the clinical diseases, understanding the types of damage sunlight causes in the DNA, recognition of DNA repair in different organisms, discovery of defective DNA repair in humans, cloning of multiple human DNA repair genes, and identification of UV-type mutations in skin cancer.

Abstract 4893: USP7 modulates UV-induced PCNA monoubiquitylation by regulating DNA polymerase eta stability

Abstract DNA polymerase eta (Polη), a product of the Xeroderma pigmentosum variant (XPV) gene, plays a unique role in translesion DNA synthesis (TLS). It is a short-lived proteasomally degraded protein and its steady-state level is tightly controlled by multiple pathways. In this study, we have identified the deubiquitylating enzyme ubiquitin-specific protease 7 (USP7) as a novel regulator of Polη stability. Polη and USP7 interact in vitro and in vivo. Overexpression of wild-type USP7, but not its interaction-defective and catalytic mutants, deubiquitylate Polη and increase its steady-state level. The data demonstrate that both physical interaction and catalytic activity of USP7 are necessary for Polη deubiquitylation and stability regulation. Interestingly, knockout of USP7 also increased the steady-state levels and slowed the turnover of both Polη and p53, which is likely caused by destabilizing MDM2, a targeting E3 ligase for ubiquitylation of both Polη and p53. Furthermore, ectopic expression of USP7 upregulated the UV-induced PCNA monoubiquitylation. In contrast, UV-induced PCNA monoubiquitylation was not observed in XPV cells ectopically expressing USP7. Taken together, our results demonstrated that USP7 facilitates UV-induced PCNA monoubiquitylation by directly and indirectly regulating Polη stability. (The work was supported by grants from NIH.) Note: This abstract was not presented at the meeting. Citation Format: Jiang Qian, Qianzhen Zhu, Kyle Pentz, Jinshan He, Qien Wang, Altaf A. Wani. USP7 modulates UV-induced PCNA monoubiquitylation by regulating DNA polymerase eta stability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4893. doi:10.1158/1538-7445.AM2014-4893

Abstract 5139: MOF cooperates with DNA polymerase eta to repair intrastrand break induced by crosslinking agents

Abstract The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Reduced levels of H4K16ac correlate with a defective DNA damage response (DDR) and double-strand break (DSB) repair post ionizing radiation exposure. MOF overexpression increases cell survival to intrastrand crosslink inducing agents like cisplatin. DNA polymerase eta (Pol H), the product of the xeroderma pigmentosum variant (XPV) gene and a Y-family DNA polymerase, plays a pivotal role in translesion DNA synthesis and was found to interact with MOF. Its interaction with MOF increases in a dose dependent manner after exposure to intrastrand DNA damage induced by cisplatin. Pol H colocalized with phosphorylated MOF and γ-H2AX foci in cisplatin treated cells. Overexpression of MOF enhances recruitment of Pol H to laser induced DNA damage. This correlated with monoubiquitination of PCNA which has been shown to facilitate the switch between the replicative DNA polymerase with the low-fidelity polymerase eta to bypass UV or cisplatin-induced DNA lesions during replication. MOF was found to interact with PCNA and regulating the ubiquitination of PCNA thereby facilitating Pol H recruitment to DNA damage sites. Note: This abstract was not presented at the meeting. Citation Format: Mayank Singh, Arun Gupta, Komal Komal, Tej K. Pandita. MOF cooperates with DNA polymerase eta to repair intrastrand break induced by crosslinking agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5139. doi:10.1158/1538-7445.AM2014-5139

Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light.

Short-wave ultraviolet light induces both mildly helix-distorting cyclobutane pyrimidine dimers (CPDs) and severely distorting (6–4) pyrimidine pyrimidone photoproducts ((6–4)PPs). The only DNA polymerase (Pol) that is known to replicate efficiently across CPDs is Polη, a member of the Y family of translesion synthesis (TLS) DNA polymerases. Phenotypes of Polη deficiency are transient, suggesting redundancy with other DNA damage tolerance pathways. Here we performed a comprehensive analysis of the temporal requirements of Y-family Pols ι and κ as backups for Polη in (i) bypassing genomic CPD and (6–4)PP lesions in vivo, (ii) suppressing DNA damage signaling, (iii) maintaining cell cycle progression and (iv) promoting cell survival, by using mouse embryonic fibroblast lines with single and combined disruptions in these Pols. The contribution of Polι is restricted to TLS at a subset of the photolesions. Polκ plays a dominant role in rescuing stalled replication forks in Polη-deficient mouse embryonic fibroblasts, both at CPDs and (6–4)PPs. This dampens DNA damage signaling and cell cycle arrest, and results in increased survival. The role of relatively error-prone Pols ι and κ as backups for Polη contributes to the understanding of the mutator phenotype of xeroderma pigmentosum variant, a syndrome caused by Polη defects.

Functional studies of human polymerase η A264P (736.6)

DNA is the blueprint which contains the information necessary for a cell to maintain normal structure and function. This essential function of DNA means the cell must necessarily preserve the integrity of its blueprint. Harmful mutations must be minimalized where they cannot be completely eliminated. One such method for maintaining the integrity of DNA is through the function of human polymerase η, a replication enzyme which gains its utility in its ability to correct thymine dimerization mutations to DNA as a result of exposure to ultraviolet radiation. Mutations in human polymerase η lead to a xeroderma pigmentosum variant (XPV) which is limited in its ability to repair damage done to DNA. This research will include introducing the A264P mutation to the catalytic core of the enzyme through site‐directed mutagenesis, transformation of the resultant DNA into E. coli cells, eventually to overexpress and purify the protein, and run translesion synthesis assays in order to determine how restricted the function of the A264P mutant is when compared to the wild‐type protein.

Xeroderma Pigmentosum: A Multidisciplinary Approach

Xeroderma pigmentosum (XP) is a rare, autosomal recessive disorder of DNA repair. Affected individuals are unable to repair ultraviolet radiation (UVR)-induced DNA damage, leading to a variety of clinical manifestations: a dramatic increase in mucocutaneous malignancies, increased lentigines, extreme photosensitivity (in approximately 50% of cases), and neurodegeneration (in approximately 30% of affected individuals). Incidence in Western Europe is recorded as 2.3 per million live births. There are eight different complementation groups, XP-A to XP-G, and XP-variant (XP-V) corresponding to the eight affected genes. Classically, XP patients were identified by clinicians for their tendency to develop severe and exaggerated sunburn on minimal sun exposure, however recently it has been shown that XP-C, XP-E and XP-V patients have normal sunburn reactions for skin type compared to the other groups, who suffer not only with severe, exaggerated sunburn, but also have an increased incidence of neurodegeneration. A diagnosis of XP should be considered in a child with either severe sunburn, increasing lentigines at exposed sites, or development of multiple skin cancers at an early age. Skin biopsy and subsequent testing in cell cultures for defective DNA repair, confirms or excludes the diagnosis. Mean life expectancy is reduced; the two main causes of mortality are skin cancer and neurodegeneration. These clinical features distinguish XP from other disorders of DNA repair, namely Trichothiodystrophy and Cockayne syndrome, although overlapping syndromes do occur. Instigation of meticulous photoprotection for all XP patients has been shown to reduce both the lentigines and number of skin cancers dramatically and would be presumed to increase life expectancy. Compliance with photoprotection is a recognised problem amongst XP patients, particularly in those without easy sunburn. This is further accentuated by lack of social acceptance for people who wear UVR-protective visors. Increased awareness of XP, both within the medical and media spheres will benefit current and future XP patients; this will aid earlier diagnosis and timely photoprotection, with better compliance, and therefore, result in an improved prognosis.

Patients with xeroderma pigmentosum complementation groups C, E and V do not have abnormal sunburn reactions

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder of DNA repair. It is divided into eight complementation groups: XP-A to XP-G (classical XP) and XP variant (XP-V). Severe and prolonged sunburn reactions on minimal sun exposure have been considered a cardinal feature of classical XP. However, it has recently become clear that not all patients have abnormal sunburn reactions. To examine sunburn reactions in a cohort of patients with XP and correlate this to the complementation group. Sixty patients with XP attending the U.K. National XP Service from 2010 to 2012 were studied. Their history of burning after minimal sun exposure was assessed using a newly developed sunburn severity score. The age at which the first skin cancer was histologically diagnosed in each patient, and the presence of any neurological abnormality, was also recorded. Sunburn severity scores were abnormally high in patients with XP-A, XP-D, XP-F and XP-G compared with non-XP controls. There was no significant difference in sunburn score of patients with XP-C, XP-E and XP-V compared with controls (P > 0·05). Patients with XP-C, XP-E and XP-V were more likely to have skin cancer diagnosed at an earlier age than those with severe sunburn on minimal sun exposure. In addition, patients with XP with severe sunburn had an increased frequency of neurological abnormalities. Not all patients with XP have a history of severe and prolonged sunburn on minimal sun exposure. The normal sunburn response of patients with XP-C, XP-E and XP-V may relate to the preservation of transcription-coupled DNA repair in these groups. Those with a history of severe sunburn on minimal sun exposure developed their first skin cancer at an older age compared with patients with XP-C, XP-E and XP-V, but they had an increased frequency of neurological abnormalities. Physicians need to be aware that about half of all patients with XP will present without a history of abnormal sunburn.

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.

PrimPol Bypasses UV Photoproducts during Eukaryotic Chromosomal DNA Replication

SummaryDNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) light-damaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol η-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells.

A novel POLH mutation causes XP-V disease and XP-V tumor proneness may involve imbalance of numerous DNA polymerases

Xeroderma pigmentosum variant (XP-V) is a subtype of xeroderma pigmentosum (XP) disease with typical pigmentation and types of cancer in the oral maxillofacial and other sun-exposed regions. Few factors of tumor proneness in XP-V have been completely elucidated with the exception of the POLH [which encodes DNA polymerase η (pol η)] mutation. The aim of the present study was to identify the POLH mutation in an XP-V patient and to explore the roles of specific additional polymerases in XP-V tumor proneness. The POLH gene was sequenced in the patient and the expression of pol η, ι, κ, θ and ζ was tested in XP-V tumor cells and cell lines, as well as in HeLa cells with POLH knockdown. The results revealed a novel, large homozygous deletion of POLH (del exon 5–9) in the patient. Lower expression of pol κ, θ and ζ were observed in the XP-V cells and similar changes were observed in HeLa cells with POLH knockdown. Consistent with XP-V tumor cells, following UV irradiation, the expression of pol κ and θ presented was significantly increased in the XP-V cell lines compared with that in the normal control cells. The unusual expression of other polymerases, besides pol η, identified in the present study indicated that these polymerases may also be key in XP-V cells genetic instability, which accelerates tumor formation.

Handling the 3-methylcytosine lesion by six human DNA polymerases members of the B-, X- and Y-families

Alkylating agents often generate 3-methylcytosine (3meC) lesions that are efficiently repaired by AlkB homologues. If AlkB homologue proteins are not functional, or the number of 3meC lesions exceeds the cellular repair capacity, the damage will persist in the genome and become substrate of DNA polymerases (Pols). Though alkylating agents are present in our environment and used in the clinics, currently nothing is known about the impact of 3meC on the accuracy and efficiency of human Pols. Here we compared the 3meC bypass properties of six human Pols belonging to the three families: B (Pol δ), X (Pols β and λ) and Y (Pols κ, ι and η). We show that under replicative conditions 3meC impairs B-family, blocks X-family, but not Y-family Pols, in particular Pols η and ι. These Pols successfully synthesize opposite 3meC; Pol ι preferentially misincorporates dTTP and Pol η dATP. The most efficient extenders from 3meC base-paired primers are Pols κ and η. Finally, using xeroderma pigmentosum variant patient cell extracts, we provide evidence that the presence of functional Pol η is mandatory to efficiently overcome 3meC by mediating complete bypass or extension. Our data suggest that Pol η is crucial for efficient 3meC bypass.

Three school‐age cases of xeroderma pigmentosum variant type

Xeroderma pigmentosum (XP) is a photosensitive genodermatosis with increased susceptibility to skin cancers. Patients are typically diagnosed with XP when they consult a dermatologist for skin cancers. The genetic analysis and 2-8 years of follow-up for three school-age patients with XP-V is described. The patients were referred to us because of increased pigmented freckles; they had not experienced abnormal sunburn or developed skin cancer at their first visit. All patients harbored a genetic mutation in the POLH gene. XPV9KO was diagnosed at age 13 with a homozygous del1661A that creates a stop codon in the non-catalytic domain of POLH. The patient practiced sun protection, effectively preventing the development of skin cancer by age 21. XPV19KO was diagnosed at age 11 with a compound heterozygous mutation of G490T and C1066T, causing POLH truncation in the catalytic domain. This patient developed basal cell carcinoma at ages 12 and 13. XPV18KO was referred to us at age 11 and diagnosed with compound heterozygous variants of c.1246_1311del66 (exon 9 skipping), a novel mutation, and c.661_764 del104 (exon 6 skipping). Freckle-like pigmentation on sun-exposed skin is sometimes the only sign of XP-V, and early diagnosis is extremely important for children.

Abstract 2029: Interactions between a selenophene compound and DNA polymerase eta.

Abstract D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-N-methylpyrrole] is a selenophene compound that has potent toxicity to various types of cancer cells but not for normal cells. More importantly, in vivo xenograft animal studies have also shown that D-501036 is a promising anticancer compound. Mechanistic studies indicated that D-501036 induces DNA double strand breaks in a dose dependent manner and blocks cell cycle progression at S-phase. D-501036 forms DNA adducts and the amount of DNA adducts increased with the concentration of D-501036. Therefore, we examined several DNA replication/repair mechanisms for their potential relationship with D-501036. Interestingly, we observed that D-501036 suppressed the expression of DNA lesion bypass polymerase η (pol η) more than 70% in both time and dose dependent manners in the ovarian Ovcar-4 cancer cells. Pol η is the key enzyme to replicate across UV induced cyclobutane pyrimidine dimers (CPD) in DNA. Genetic defects in the pol η encoding gene result in a disease called Xeroderma Pigmentosum variant (XP-V). In addition to its biological function, biochemical studies have shown that pol η can also efficiently bypass cisplatin intrastrand crosslinked guanosines (Pt-GG). In consistence with the biochemical studies, cellular studies have shown that the expression of pol η reduces the cytotoxicity of cisplatin in Ovcar-4 cells. These results suggested that pol η plays an important role in modulating the cellular sensitivity to cisplatin. Although cisplatin is an important therapeutic agent for the treatment of ovarian cancer but the development of resistance against cisplatin has been a major drawback. Given that D-501036 suppresses the expression of pol η, we performed a cytotoxicity study using the combination of D-501036 and cisplatin. Our results indicated that D-501036 can potentiate the sensitivity of Ovcar-4 cells to cisplatin more than 1,000- fold. Together, our results suggested that D-501036 is a promising compound that can be used to enhance the activity of cisplatin for the treatment of ovarian cancer. Further studies are undergoing to determine the mechanisms of action of D-501036, the information obtained will facilitate the development of new compounds to improve the treatment for ovarian cancer. Citation Format: Anne Rietz, Yih-wen Chen, Kai-Ming Chou. Interactions between a selenophene compound and DNA polymerase eta. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2029. doi:10.1158/1538-7445.AM2013-2029

Site‐directed mutagenesis, expression, purification and translesion synthesis analysis of human DNA polymerase η mutations found in xeroderma pigmentosum variant and melanoma patients

The variant form of human syndrome Xeroderma Pigmentosum (XP‐V) is caused by mutations in DNA polymerase η (hpolη). XP‐V patients are deficient in repairing the DNA damage caused by ultraviolet (UV) light in the form of cyclobutane‐pyrimidine dimers (CPD). Many missense mutations in hpolη catalytic core (1–432aa) have been identified in XP‐V patients, and mutations have also been implicated in Melanoma. The aim of current research is to confirm that the catalytic core of hpolη is 1–432aa. Hpolη (1–432aa) has been constructed. It will be expressed in E.coli and tested in primer extension assays to assess its effectiveness during translesion synthesis (TLS). Several of the point mutations have been successfully introduced in to hpolη (1–432aa) by site‐directed mutagenesis. Those mutants are expressed in E.coli cells and will be examined for their activity during translesion synthesis (TLS). Source of research support: Student research grant to Mukesh Kumar, Department of Chemistry, TTU, Cookeville, TN, Faculty grant to Xiaohua Jiang, College of Arts and Sciences, TTU, Cookeville, TN

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.

The Role of Altered Nucleotide Excision Repair and UVB-Induced DNA Damage in Melanomagenesis

UVB radiation is the most mutagenic component of the UV spectrum that reaches the earth’s surface and causes the development of DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts. UV radiation usually results in cellular death, but if left unchecked, it can affect DNA integrity, cell and tissue homeostasis and cause mutations in oncogenes and tumour-suppressor genes. These mutations, if unrepaired, can lead to abnormal cell growth, increasing the risk of cancer development. Epidemiological data strongly associates UV exposure as a major factor in melanoma development, but the exact biological mechanisms involved in this process are yet to be fully elucidated. The nucleotide excision repair (NER) pathway is responsible for the repair of UV-induced lesions. Patients with the genetic disorder Xeroderma Pigmentosum have a mutation in one of eight NER genes associated with the XP complementation groups XP-A to XP-G and XP variant (XP-V). XP is characterized by diminished repair capacity, as well as a 1000-fold increase in the incidence of skin cancers, including melanoma. This has suggested a significant role for NER in melanoma development as a result of UVB exposure. This review discusses the current research surrounding UVB radiation and NER capacity and how further investigation of NER could elucidate the role of NER in avoiding UV-induced cellular death resulting in melanomagenesis.

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.

Ancient origin of a Japanese xeroderma pigmentosum founder mutation

Ancient origin of a Japanese xeroderma pigmentosum founder mutation

Regulation of the specialized DNA polymerase eta: Revisiting the biological relevance of its PCNA‐ and ubiquitin‐binding motifs

During translesion synthesis (TLS), low-fidelity polymerases of the Y-family polymerases bypass DNA damages that block the progression of conventional processive DNA polymerases, thereby allowing the completion of DNA replication. Among the TLS polymerases, DNA polymerase eta (polη) performs nucleotide incorporation past ultraviolet (UV) photoproducts and is deficient in cancer-prone xeroderma pigmentosum variant (XPV) syndrome. Upon UV irradiation, the DNA sliding clamp PCNA is monoubiquitylated on its conserved Lys-164. This event is considered to facilitate the TLS process in vivo since polη preferentially interacts with monoubiquitylated PCNA through its ubiquitin-binding domain (UBZ) as well as its PCNA interacting peptide (PIP)-box. However, recent observations questioned this model. Therefore, in this study, we re-examined the relative contribution of the regulatory UBZ and PIP domains of polη in response to UVC. We show that simultaneous invalidation of both motifs confers sensitivity to UVC, sensitization by low concentrations of caffeine, prolonged inhibition of DNA synthesis and persistent S phase checkpoint activation, all characteristic features of XPV cells. While each domain is essential for efficient accumulation of polη in replication factories, mutational inactivation of UBZ or PIP motif only confers a slight sensitivity to UVC indicating that, although informative, polη focus analysis is not a reliable tool to assess the polη's ability to function in TLS in vivo. Taken together, these data indicate that PIP and UBZ motifs are not required for recruitment but for retention of polη at sites of stalled replication forks. We propose that this is a way to ensure that a sufficient amount of the protein is available for its bypass function.

Down-regulation of Polη expression leads to increased DNA damage, apoptosis and enhanced S phase arrest in L-02 cells exposed to hydroquinone

DNA polymerase eta (Polη), the product of the xeroderma pigmentosum variant gene, is required for translesion DNA synthesis, and plays a pivotal role in preventing genome instability after DNA damage induced by genotoxic agents. Studies have previously suggested a link between Polη and susceptibility to hydroquinone (HQ)-induced toxicity. To further address the role of Polη in the response of L-02 cells to HQ, we employed RNA interference to silence Polη expression in L-02 cells and examined the susceptibility of these Polη-deficient cells to the toxic effects of HQ. In this study, cell survival rate was determined using the MTT assay, DNA damage was determined by the Comet assay, apoptosis and cell cycle distribution were determined using flow cytometry, the mRNA expression levels of Polη were determined by real-time PCR, and the protein expression levels of Polη and γ-H2AX were determined by Western blot, γ-H2AX foci were visualized by confocal laser scanning fluorescence microscopy after cells were exposed to HQ at various concentrations for 24h in vitro. The results showed that stable Polη-knockdown cells were successfully constructed and more than 80% inhibition of Polη expression was confirmed. The results also showed that down-regulation of Polη led to a decrease in cell proliferation and an enhanced susceptibility to HQ-induced cytotoxicity. Polη-deficient cells were 2-fold more sensitive to HQ when compared with nonspecific siRNA control cells. Moreover, Polη-silenced L-02 cells treated with HQ displayed an increased level of DNA double-strand breaks as measured by olive tail moment, and an elevated DNA damage response as indicated by the induction of γ-H2AX. In addition, knockdown of Polη resulted in more enhanced apoptosis and more pronounced S phase arrest following HQ treatment. Together, these results show that Polη plays an important role in the response of L-02 cells to HQ-induced DNA damage.