Detection of endogenous translesion DNA synthesis in single mammalian cells.

Translesion DNA synthesis (TLS) is an evolutionarily conserved process that cells activate to tolerate DNA damage. TLS facilitates proliferation under DNA damage conditions and is exploited by cancer cells to gain therapy resistance. It has been so far challenging to analyze endogenous TLS factors such as PCNAmUb and TLS DNA polymerases in single mammalian cells due to a lack of suitable detection tools. We have adapted a flow cytometry-based quantitative method allowing detection of endogenous, chromatin-bound TLS factors in single mammalian cells, either untreated or exposed to DNA-damaging agents. This high-throughput procedure is quantitative, accurate, and allows unbiased analysis of TLS factors' recruitment to chromatin, as well as occurrence of DNA lesions with respect to the cell cycle. We also demonstrate detection of endogenous TLS factors by immunofluorescence microscopy and provide insights into TLS dynamics upon DNA replication forks stalled by UV-C-induced DNA damage.

Nontraditional Roles of DNA Polymerase Eta Support Genome Duplication and Stability.

DNA polymerase eta (Pol η) is a Y-family polymerase and the product of the POLH gene. Autosomal recessive inheritance of POLH mutations is the cause of the xeroderma pigmentosum variant, a cancer predisposition syndrome. This review summarizes mounting evidence for expanded Pol η cellular functions in addition to DNA lesion bypass that are critical for maintaining genome stability. In vitro, Pol η displays efficient DNA synthesis through difficult-to-replicate sequences, catalyzes D-loop extensions, and utilizes RNA-DNA hybrid templates. Human Pol η is constitutively present at the replication fork. In response to replication stress, Pol η is upregulated at the transcriptional and protein levels, and post-translational modifications regulate its localization to chromatin. Numerous studies show that Pol η is required for efficient common fragile site replication and stability. Additionally, Pol η can be recruited to stalled replication forks through protein-protein interactions, suggesting a broader role in replication fork recovery. During somatic hypermutations, Pol η is recruited by mismatch repair proteins and is essential for VH gene A:T basepair mutagenesis. Within the global context of repeat-dense genomes, the recruitment of Pol η to perform specialized functions during replication could promote genome stability by interrupting pure repeat arrays with base substitutions. Alternatively, not engaging Pol η in genome duplication is costly, as the absence of Pol η leads to incomplete replication and increased chromosomal instability.

DNA polymerase eta protects human cells against DNA damage induced by the tumor chemotherapeutic temozolomide.

Human DNA polymerases can bypass DNA lesions performing translesion synthesis (TLS), a mechanism of DNA damage tolerance. Tumor cells use this mechanism to survive lesions caused by specific chemotherapeutic agents, resulting in treatment relapse. Moreover, TLS polymerases are error-prone and, thus, can lead to mutagenesis, increasing the resistance potential of tumor cells. DNA polymerase eta (pol eta) - a key protein from this group - is responsible for protecting against sunlight-induced tumors. Xeroderma Pigmentosum Variant (XP-V) patients are deficient in pol eta activity, which leads to symptoms related to higher sensitivity and increased incidence of skin cancer. Temozolomide (TMZ) is a chemotherapeutic agent used in glioblastoma and melanoma treatment. TMZ damages cells' genomes, but little is known about the role of TLS in TMZ-induced DNA lesions. This work investigates the effects of TMZ treatment in human XP-V cells, which lack pol eta, and in its complemented counterpart (XP-V comp). Interestingly, TMZ reduces the viability of XP-V cells compared to TLS proficient control cells. Furthermore, XP-V cells treated with TMZ presented increased phosphorylation of H2AX, forming γH2AX, compared to control cells. However, cell cycle assays indicate that XP-V cells treated with TMZ replicate damaged DNA and pass-through S-phase, arresting in the G2/M-phase. DNA fiber assay also fails to show any specific effect of TMZ-induced DNA damage blocking DNA elongation in pol eta deficient cells. These results show that pol eta plays a role in protecting human cells from TMZ-induced DNA damage, but this can be different from its canonical TLS mechanism. The new role opens novel therapeutic possibilities of using pol eta as a target to improve the efficacy of TMZ-based therapies against cancer.

miR-20b downregulates polymerases κ and θ in XP-V tumor cells.

XP-V is a subtype of Xeroderma pigmentosum diseases with typical pigmentation and cancers in sun-exposed regions. The present study investigated the role of microRNA-20b (miR-20b) in the imbalance of polymerase expression levels in XP-V tumor cells. Following software prediction results, certain miRNAs were chosen as candidate regulators for the observed imbalance in polymerases in XP-V tumor cells. Reverse transcription-quantitative polymerase chain reaction and western blot were used to test candidate miRNAs for their ability to reduce the expression of these polymerases. A luciferase reporter assay was used to further verify the western blot results. Polymerases κ and θ were expressed at lower levels in XP-V tumor cells compared to normal control cells. A positive correlation was demonstrated between miR-20b and polymerases κ and θ. It was also demonstrated that a proportion of miRNAs had no effect on polymerases κ and θ, despite the software predicting that these miRNAs would target these two polymerases. Therefore, miR-20b may be responsible for the low expression levels of polymerase κ and θ in XP-V tumor cells, which accelerated mismatch in DNA replication repairing.

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.

UV-induced mutations in epidermal cells of mice defective in DNA polymerase η and/or ι.

Xeroderma pigmentosum variant (XP-V) is a human rare inherited recessive disease, predisposed to sunlight-induced skin cancer, which is caused by deficiency in DNA polymerase η (Polη). Polη catalyzes accurate translesion synthesis (TLS) past pyrimidine dimers, the most prominent UV-induced lesions. DNA polymerase ι (Polι) is a paralog of Polη that has been suggested to participate in TLS past UV-induced lesions, but its function in vivo remains uncertain. We have previously reported that Polη-deficient and Polη/Polι double-deficient mice showed increased susceptibility to UV-induced carcinogenesis. Here, we investigated UV-induced mutation frequencies and spectra in the epidermal cells of Polη- and/or Polι-deficient mice. While Polη-deficient mice showed significantly higher UV-induced mutation frequencies than wild-type mice, Polι deficiency did not influence the frequencies in the presence of Polη. Interestingly, the frequencies in Polη/Polι double-deficient mice were statistically lower than those in Polη-deficient mice, although they were still higher than those of wild-type mice. Sequence analysis revealed that most of the UV-induced mutations in Polη-deficient and Polη/Polι double-deficient mice were base substitutions at dipyrimidine sites. An increase in UV-induced mutations at both G:C and A:T pairs associated with Polη deficiency suggests that Polη contributes to accurate TLS past both thymine- and cytosine-containing dimers in vivo. A significant decrease in G:C to A:T transition in Polη/Polι double-deficient mice when compared with Polη-deficient mice suggests that Polι is involved in error-prone TLS past cytosine-containing dimers when Polη is inactivated.

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.

Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage.

How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.