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The Functions of Serine 687 Phosphorylation of Human DNA Polymerase η in UV Damage Tolerance.

ABSTRACT: DNA polymerase η (polη) is a Y-family translesion synthesis polymerase that plays a key role in the cellular tolerance toward UV irradiation-induced DNA damage. Here, we identified, for the first time, the phosphorylation of serine 687 (Ser(687)), which is located in the highly conserved nuclear localization signal (NLS) region of human polη and is mediated by cyclin-dependent kinase 2 (CDK2). We also showed that this phosphorylation is stimulated in human cells upon UV light exposure and results in diminished interaction of polη with proliferating cell nuclear antigen (PCNA). Furthermore, we demonstrated that the phosphorylation of Ser(687) in polη confers cellular protection from UV irradiation and increases the efficiency in replication across a site-specifically incorporated cyclobutane pyrimidine dimer in human cells. Based on these results, we proposed a mechanistic model where Ser(687) phosphorylation functions in the reverse polymerase switching step of translesion synthesis: The phosphorylation brings negative charges to the NLS of polη, which facilitates its departure from PCNA, thereby resetting the replication fork for highly accurate and processive DNA replication. Thus, our study, together with previous findings, supported that the posttranslational modifications of NLS of polη played a dual role in polymerase switching, where Lys(682) deubiquitination promotes the recruitment of polη to PCNA immediately prior to lesion bypass and Ser(687) phosphorylation stimulates its departure from the replication fork immediately after lesion bypass.

SUBMITTER: Dai X

PROVIDER: S-EPMC5083098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The Functions of Serine 687 Phosphorylation of Human DNA Polymerase eta in UV Damage Tolerance

Project description:DNA polymerase (pol) is a Y-family translesion synthesis polymerase that plays a key role in the cellular tolerance toward UV irradiation-induced DNA damage. Here, we identified, for the first time, the phosphorylation of serine 687 (S687), which islocated in the highly conserved nuclear localization signal (NLS) region of human pol and is mediated by cyclin-dependent kinase 2 (CDK2). We also showed that this phosphorylation is stimulated in human cells upon UV light exposure and results in diminished interaction of pol with proliferating cell nuclear antigen (PCNA). Furthermore, we demonstrated that the phosphorylation of S687 in polconfers cellular protection fromUV irradiation and increases the efficiency in replication across a site-specifically incorporated cyclobutane pyrimidine dimer in human cells. Based on these results, we proposed a mechanistic model where S687 phosphorylation functions in the reverse polymerase switching step of translesion synthesis: The phosphorylation brings negative charges to the NLS of pol, which facilitates its departure from PCNA, thereby resetting the replication fork for highly accurate and processive DNA replication. Thus, our study, together with previous findings, supported that the post-translational modifications of NLS of pol played a dual role in polymerase switching, where K682 deubiquitination promotes the recruitment of pol to PCNA immediately prior to lesion bypass and S687 phosphorylation stimulates its departure from the replication fork immediately after lesion bypass.

2016-07-19 | PXD002946 | Pride

Multisite SUMOylation restrains DNA polymerase η interactions with DNA damage sites.

Project description:Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.

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USP7 modulates UV-induced PCNA monoubiquitination by regulating DNA polymerase eta stability.

Project description:DNA polymerase eta (Pol?) has unique and pivotal functions in several DNA damage-tolerance pathways. Steady-state level of this short-lived protein is tightly controlled by multiple mechanisms including proteolysis. Here, we have identified the deubiquitinating enzyme (DUB), ubiquitin-specific protease 7 (USP7), as a novel regulator of Pol? stability. USP7 regulates Pol? stability through both indirect and direct mechanisms. Knockout of USP7 increased the steady-state level of Pol? and slowed down the turnover of both Pol? and p53 proteins through destabilizing their E3 ligase murine double minute 2 (Mdm2). Also, USP7 physically binds Pol? in vitro and in vivo. Overexpression of wild-type USP7 but not its catalytically-defective mutants deubiquitinates Pol? and increases its cellular steady-state level. Thus, USP7 directly serves as a specific DUB for Pol?. Furthermore, ectopic expression of USP7 promoted the UV-induced proliferating cell nuclear antigen (PCNA) monoubiquitination in Pol?-proficient but not in Pol?-deficient XPV (Xeroderma pigmentosum variant) cells, suggesting that USP7 facilitates UV-induced PCNA monoubiquitination by stabilizing Pol?. Taken together, our findings reveal a modulatory role of USP7 in PCNA ubiquitination-mediated stress-tolerance pathways by fine-tuning Pol? turnover.

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Structural and mechanistic studies of polymerase η bypass of phenanthriplatin DNA damage.

Project description:Platinum drugs are a mainstay of anticancer chemotherapy. Nevertheless, tumors often display inherent or acquired resistance to platinum-based treatments, prompting the search for new compounds that do not exhibit cross-resistance with current therapies. Phenanthriplatin, cis-diamminephenanthridinechloroplatinum(II), is a potent monofunctional platinum complex that displays a spectrum of activity distinct from those of the clinically approved platinum drugs. Inhibition of RNA polymerases by phenanthriplatin lesions has been implicated in its mechanism of action. The present study evaluates the ability of phenanthriplatin lesions to inhibit DNA replication, a function disrupted by traditional platinum drugs. Phenanthriplatin lesions effectively inhibit DNA polymerases ν, ζ, and κ and the Klenow fragment. In contrast to results obtained with DNA damaged by cisplatin, all of these polymerases were capable of inserting a base opposite a phenanthriplatin lesion, but only Pol η, an enzyme efficient in translesion synthesis, was able to fully bypass the adduct, albeit with low efficiency. X-ray structural characterization of Pol η complexed with site-specifically platinated DNA at both the insertion and +1 extension steps reveals that phenanthriplatin on DNA interacts with and inhibits Pol η in a manner distinct from that of cisplatin-DNA adducts. Unlike cisplatin and oxaliplatin, the efficacies of which are influenced by Pol η expression, phenanthriplatin is highly toxic to both Pol η+ and Pol η- cells. Given that increased expression of Pol η is a known mechanism by which cells resist cisplatin treatment, phenanthriplatin may be valuable in the treatment of cancers that are, or can easily become, resistant to cisplatin.

| S-EPMC4078841 | biostudies-literature

Regulated proteolysis of DNA polymerase eta during the DNA-damage response in C. elegans.

Project description:Both the POLH-1 (pol eta) translesion synthesis (TLS) DNA polymerase and the GEI-17 SUMO E3 ligase are essential for the efficient replication of damaged chromosomes in Caenorhabditis elegans embryos. Here we study how POLH-1 is regulated during a DNA-damage response in these embryos. We report that DNA damage triggers the degradation of POLH-1 and that degradation is mediated by the Cul4-Ddb1-Cdt2 (CRL4-Cdt2) pathway that has previously been shown to degrade the replication factor Cdt1 during S phase. We also show that GEI-17 protects POLH-1 from CRL4-Cdt2-mediated destruction until after it has performed its function in TLS, and this is likely via SUMOylation of POLH-1. These studies reveal that POLH-1 undergoes DNA-damage-induced proteolysis and that GEI-17 regulates the timing of this proteolysis. Implications for how this system may control the removal of POLH-1 from replication forks after TLS are discussed.

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UV-B radiation induces epithelial tumors in mice lacking DNA polymerase eta and mesenchymal tumors in mice deficient for DNA polymerase iota.

Project description:DNA polymerase eta (Pol eta) is the product of the Polh gene, which is responsible for the group variant of xeroderma pigmentosum, a rare inherited recessive disease which is characterized by susceptibility to sunlight-induced skin cancer. We recently reported in a study of Polh mutant mice that Pol eta is involved in the somatic hypermutation of immunoglobulin genes, but the cancer predisposition of Polh-/- mice has not been examined until very recently. Another translesion synthesis polymerase, Pol iota, a Pol eta paralog encoded by the Poli gene, is naturally deficient in the 129 mouse strain, and the function of Pol iota is enigmatic. Here, we generated Polh Poli double-deficient mice and compared the tumor susceptibility of them with Polh- or Poli-deficient animals under the same genetic background. While Pol iota deficiency does not influence the UV sensitivity of mouse fibroblasts irrespective of Polh genotype, Polh Poli double-deficient mice show slightly earlier onset of skin tumor formation. Intriguingly, histological diagnosis after chronic treatment with UV light reveals that Pol iota deficiency leads to the formation of mesenchymal tumors, such as sarcomas, that are not observed in Polh(-/-) mice. These results suggest the involvement of the Pol eta and Pol iota proteins in UV-induced skin carcinogenesis.

| S-EPMC1636855 | biostudies-literature

DNA polymerase η modulates replication fork progression and DNA damage responses in platinum-treated human cells.

Project description:Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.

| S-EPMC6505966 | biostudies-literature

Spatiotemporal recruitment of human DNA polymerase delta to sites of UV damage.

Project description:Human DNA polymerase ? (Pol ?) is involved in various DNA damage responses in addition to its central role in DNA replication. The Pol ?4 holoenzyme consists of four subunits, p125, p50, p68 and p12. It has been established that the p12 subunit is rapidly degraded in response to DNA damage by UV leading to the in vivo conversion of Pol ?4 to Pol ?3, a trimeric form lacking the p12 subunit. We provide the first analysis of the time-dependent recruitment of the individual Pol ? subunits to sites of DNA damage produced by UV irradiation through 5 ?m polycarbonate filters by immunofluorescence microscopy and laser scanning cytometry (LSC). Quantitative analysis demonstrates that the recruitments of the three large subunits was near complete by 2 h and did not change significantly up to 4 h after UV exposure. However, the recruitment of p12 was incomplete even at 4 h, with about 70% of the Pol ? lacking the p12 subunit. ChIP analysis of Pol ? after global UV irradiation further demonstrates that only p125, p50 and p68 were present. Thus, Pol ?3 is the predominant form of Pol ? at sites of UV damage as a result of p12 degradation. Using LSC, we have further confirmed that Pol ? was recruited to CPD damage sites in all phases of the cell cycle. Collectively, our results show that Pol ? at the DNA damage site is the Pol ? trimer lacking p12 regardless of the cell cycle phase.

| S-EPMC3419061 | biostudies-literature

Rev1 promotes replication through UV lesions in conjunction with DNA polymerases η, ι, and κ but not DNA polymerase ζ.

Project description:Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans.

| S-EPMC4699387 | biostudies-literature

DNA polymerase η contributes to lagging strand synthesis

Project description:DNA polymerase eta (pol eta) is best known for its ability to bypass UV-induced thymine-thymine (T-T) dimers and other bulky DNA lesions, but pol eta also has other cellular roles. Here, we present evidence that pol eta competes with DNA polymerases alpha and delta for the synthesis of the lagging strand genome-wide, where it also shows a preference for T-T in the DNA template. Moreover, we found that the C-terminus of pol eta which contains a PCNA-Interacting Protein motif is required for pol eta to function in lagging strand synthesis. Finally, we provide evidence that a pol η dependent signature is also found to be lagging strand specific in patients with skin cancer. Taken together, these findings provide insight into the physiological role of DNA synthesis by pol eta and have implications for our understanding of how our genome is replicated to avoid mutagenesis, genome instability and cancer.

2018-12-14 | GSE110241 | GEO

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