Pold4 subunit of replicative polymerase δ promotes fork slowing at broken templates.

Single-strand breaks (SSBs) are the most frequent type of lesion, and replication across such lesions leads to double-strand breaks (DSBs). DSBs that arise during replication are repaired by homologous recombination (HR) and are suppressed by fork reversal. Poly[ADP-ribose] polymerase I (PARP1) and the proofreading exonuclease activity of replicative polymerase ε (Polε) are required for fork reversal when leading strand replication encounters SSBs. However, the mechanism underlying fork reversal at the SSB during lagging-strand replication remains elusive. We here demonstrate that the Pold4 subunit of replicative polymerase δ (Polδ) plays a role in promoting fork reversal during lagging strand replication on a broken template. POLD4-/- cells exhibited heightened sensitivity to camptothecin (CPT) but not to other DNA-damaging agents compared to wild-type cells. This selective CPT sensitivity in POLD4-/- cells suggests that Pold4 suppresses DSBs during replication, as CPT induces significant SSBs during replication, which subsequently lead to DSBs. To explore the functional interactions among Pold4, Polε exonuclease, and PARP1 in DSB suppression, we generated PARP1-/-, POLD4-/-, Polε exonuclease-deficient POLE1exo-/-, PARP1-/-/POLD4-/-, and POLD4-/-/POLE1exo-/- cells. These epistasis analyses showed that Pold4 is involved in the PARP1-Polε exonuclease-mediated fork reversal following CPT treatment. These results suggest that Pold4 aids in fork reversal when lagging strand replication stalls on a broken template. In conclusion, the Pold4 subunit of Polδ has roles in the PARP1-Polε exonuclease-mediated fork reversal, contributing to the suppression of DSBs.

Dominant roles of BRCA1 in cellular tolerance to a chain-terminating nucleoside analog, alovudine.

Alovudine is a chain-terminating nucleoside analog (CTNA) that is frequently used as an antiviral and anticancer agent. Generally, CTNAs inhibit DNA replication after their incorporation into nascent DNA during DNA synthesis by suppressing subsequent polymerization, which restricts the proliferation of viruses and cancer cells. Alovudine is a thymidine analog used as an antiviral drug. However, the mechanisms underlying the removal of alovudine and DNA damage tolerance pathways involved in cellular resistance to alovudine remain unclear. Here, we explored the DNA damage tolerance pathways responsible for cellular tolerance to alovudine and found that BRCA1-deficient cells exhibited the highest sensitivity to alovudine. Moreover, alovudine interfered with DNA replication in two distinct mechanisms: first: alovudine incorporated at the end of nascent DNA interfered with subsequent DNA synthesis; second: DNA replication stalled on the alovudine-incorporated template strand. Additionally, BRCA1 facilitated the removal of the incorporated alovudine from nascent DNA, and BRCA1-mediated homologous recombination (HR) contributed to the progressive replication on the alovudine-incorporated template. Thus, we have elucidated the previously unappreciated mechanism of alovudine-mediated inhibition of DNA replication and the role of BRCA1 in cellular tolerance to alovudine.

Metabolic stress-induced long ncRNA transcription governs the formation of meiotic DNA breaks in the fission yeast fbp1 gene.

Meiotic recombination is a pivotal process that ensures faithful chromosome segregation and contributes to the generation of genetic diversity in offspring, which is initiated by the formation of double-strand breaks (DSBs). The distribution of meiotic DSBs is not uniform and is clustered at hotspots, which can be affected by environmental conditions. Here, we show that non-coding RNA (ncRNA) transcription creates meiotic DSBs through local chromatin remodeling in the fission yeast fbp1 gene. The fbp1 gene is activated upon glucose starvation stress, in which a cascade of ncRNA-transcription in the fbp1 upstream region converts the chromatin configuration into an open structure, leading to the subsequent binding of transcription factors. We examined the distribution of meiotic DSBs around the fbp1 upstream region in the presence and absence of glucose and observed several new DSBs after chromatin conversion under glucose starvation conditions. Moreover, these DSBs disappeared when cis-elements required for ncRNA transcription were mutated. These results indicate that ncRNA transcription creates meiotic DSBs in response to stress conditions in the fbp1 upstream region. This study addressed part of a long-standing unresolved mechanism underlying meiotic recombination plasticity in response to environmental fluctuation.

Homology recognition without double-stranded DNA-strand separation in D-loop formation by RecA.

RecA protein and RecA/Rad51 orthologues are required for homologous recombination and DNA repair in all living creatures. RecA/Rad51 catalyzes formation of the D-loop, an obligatory recombination intermediate, through an ATP-dependent reaction consisting of two phases: homology recognition between double-stranded (ds)DNA and single-stranded (ss)DNA to form a hybrid-duplex core of 6-8 base pairs and subsequent hybrid-duplex/D-loop processing. How dsDNA recognizes homologous ssDNA is controversial. The aromatic residue at the tip of the ß-hairpin loop (L2) was shown to stabilize dsDNA-strand separation. We tested a model in which dsDNA strands were separated by the aromatic residue before homology recognition and found that the aromatic residue was not essential to homology recognition, but was required for D-loop processing. Contrary to the model, we found that the double helix was not unwound even a single turn during search for sequence homology, but rather was unwound only after the homologous sequence was recognized. These results suggest that dsDNA recognizes its homologous ssDNA before strand separation. The search for homologous sequence with homologous ssDNA without dsDNA-strand separation does not generate stress within the dsDNA; this would be an advantage for dsDNA to express homology-dependent functions in vivo and also in vitro.

The proofreading exonuclease of leading-strand DNA polymerase epsilon prevents replication fork collapse at broken template strands.

Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo-/-, CTF18-/-, PARP1-/-, CTF18-/-/POLE1exo-/-, PARP1-/-/POLE1exo-/-, and CTF18-/-/PARP1-/- cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo-/- and HDR-deficient BRCA1-/- cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo-/- with BRCA1-/- mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.

PCNA recruits cohesin loader Scc2 to ensure sister chromatid cohesion.

Sister chromatid cohesion, established during replication by the ring-shaped multiprotein complex cohesin, is essential for faithful chromosome segregation. Replisome-associated proteins are required to generate cohesion by two independent pathways. One mediates conversion of cohesins bound to unreplicated DNA ahead of replication forks into cohesive entities behind them, while the second promotes cohesin de novo loading onto newly replicated DNA. The latter process depends on the cohesin loader Scc2 (NIPBL in vertebrates) and the alternative PCNA loader CTF18-RFC. However, the mechanism of de novo cohesin loading during replication is unknown. Here we show that PCNA physically recruits the yeast cohesin loader Scc2 via its C-terminal PCNA-interacting protein motif. Binding to PCNA is crucial, as the scc2-pip mutant deficient in Scc2-PCNA interaction is defective in cohesion when combined with replisome mutants of the cohesin conversion pathway. Importantly, the role of NIPBL recruitment to PCNA for cohesion generation is conserved in vertebrate cells.

CTF18-RFC contributes to cellular tolerance against chain-terminating nucleoside analogs (CTNAs) in cooperation with proofreading exonuclease activity of DNA polymerase ε.

Chemotherapeutic nucleoside analogs, such as cytarabine (Ara-C), are incorporated into genomic DNA during replication. Incorporated Ara-CMP (Ara-cytidine monophosphate) serves as a chain terminator and inhibits DNA synthesis by replicative polymerase epsilon (Polε). The proofreading exonuclease activity of Polε removes the misincorporated Ara-CMP, thereby contributing to the cellular tolerance to Ara-C. Purified Polε performs proofreading, and it is generally believed that proofreading in vivo does not need additional factors. In this study, we demonstrated that the proofreading by Polε in vivo requires CTF18, a component of the leading-strand replisome. We found that loss of CTF18 in chicken DT40 cells and human TK6 cells results in hypersensitivity to Ara-C, indicating the conserved function of CTF18 in the cellular tolerance of Ara-C. Strikingly, we found that proofreading-deficient POLE1D269A/-, CTF18-/-, and POLE1D269A/-/CTF18-/- cells showed indistinguishable phenotypes, including the extent of hypersensitivity to Ara-C and decreased replication rate with Ara-C. This observed epistatic relationship between POLE1D269A/- and CTF18-/- suggests that they are interdependent in removing mis-incorporated Ara-CMP from the 3' end of primers. Mechanistically, we found that CTF18-/- cells have reduced levels of chromatin-bound Polε upon Ara-C treatment, suggesting that CTF18 contributes to the tethering of Polε on fork at the stalled end and thereby facilitating the removal of inserted Ara-C. Collectively, these data reveal the previously unappreciated role of CTF18 in Polε-exonuclease-mediated maintenance of the replication fork upon Ara-C incorporation.

Application of neural network-based image analysis to detect sister chromatid cohesion defects.

Sister chromatid cohesion (SCC) is mediated by the cohesin complex and its regulatory proteins. To evaluate the involvement of a protein in cohesin regulation, preparations of metaphase chromosome spreads and classifications of chromosome shapes after depletion of the target protein are commonly employed. Although this is a convenient and approved method, the evaluation and classification of each chromosome shape has to be performed manually by researchers. Therefore, this method is time consuming, and the results might be affected by the subjectivity of researchers. In this study, we developed neural network-based image recognition models to judge the positional relationship of sister chromatids, and thereby detect SCC defects. Transfer learning models based on SqueeezeNet or ResNet-18 were trained with more than 600 chromosome images labeled with the type of chromosome, which were classified according to the positional relationship between sister chromatids. The SqueezeNet-based trained model achieved a concordance rate of 73.1% with the sample answers given by a researcher. Importantly, the model successfully detected the SCC defect in the CTF18 deficient cell line, which was used as an SCC-defective model. These results indicate that neural-network-based image recognition models are valuable tools for examining SCC defects in different genetic backgrounds.

Lestaurtinib induces DNA damage that is related to estrogen receptor activation.

A number of chemicals in the environment pose a threat to human health. Recent studies indicate estradiol induces DNA damage through the activation of the estrogen receptor alpha (ERα). Given that many environmental chemical compounds act like hormones once they enter the human body, it is possible that they induce DNA damage in the same way as estradiol, which is of great concern to females with the BRCA1 mutation. In this study, we developed an antibody-based high content method measuring γH2AX, a biomarker for DNA damage, to test a subset of 907 chemical compounds in MCF7 cells. The assay was optimized for a 1536 well plate format and had a satisfactory assay performance with Z-factor of 0.67. From the screening, we identified 128 compounds that induce γH2AX expression in the cells. These compounds were further examined for their γH2AX induction in the presence of an ER inhibitor, tamoxifen. After tamoxifen treatment, four compounds induced less γH2AX expression compared to those without tamoxifen treatment, suggesting these compounds induced γH2AX that is related to ERα activation. These four compounds were chosen for further studies to assess their ERα activating capability and c-MYC induction. Only lestaurtinib, a selective tyrosine kinase inhibitor, induced ERα activation, which was confirmed by both ERα beta-lactamase reporter gene assay and molecular docking analysis. Lestaurtinib also increased c-MYC expression, a target gene of ERα signaling, measured by the quantitative PCR method. This data suggests that lestaurtinib acts as a DNA damage inducer that is related to ERα activation.

Regulation Mechanisms of Meiotic Recombination Revealed from the Analysis of a Fission Yeast Recombination Hotspot ade6-M26.

Meiotic recombination is a pivotal event that ensures faithful chromosome segregation and creates genetic diversity in gametes. Meiotic recombination is initiated by programmed double-strand breaks (DSBs), which are catalyzed by the conserved Spo11 protein. Spo11 is an enzyme with structural similarity to topoisomerase II and induces DSBs through the nucleophilic attack of the phosphodiester bond by the hydroxy group of its tyrosine (Tyr) catalytic residue. DSBs caused by Spo11 are repaired by homologous recombination using homologous chromosomes as donors, resulting in crossovers/chiasmata, which ensure physical contact between homologous chromosomes. Thus, the site of meiotic recombination is determined by the site of the induced DSB on the chromosome. Meiotic recombination is not uniformly induced, and sites showing high recombination rates are referred to as recombination hotspots. In fission yeast, ade6-M26, a nonsense point mutation of ade6 is a well-characterized meiotic recombination hotspot caused by the heptanucleotide sequence 5'-ATGACGT-3' at the M26 mutation point. In this review, we summarize the meiotic recombination mechanisms revealed by the analysis of the fission ade6-M26 gene as a model system.

Multi-Layered Regulations on the Chromatin Architectures: Establishing the Tight and Specific Responses of Fission Yeast fbp1 Gene Transcription.

Transcriptional regulation is pivotal for all living organisms and is required for adequate response to environmental fluctuations and intercellular signaling molecules. For precise regulation of transcription, cells have evolved regulatory systems on the genome architecture, including the chromosome higher-order structure (e.g., chromatin loops), location of transcription factor (TF)-binding sequences, non-coding RNA (ncRNA) transcription, chromatin configuration (e.g., nucleosome positioning and histone modifications), and the topological state of the DNA double helix. To understand how these genome-chromatin architectures and their regulators establish tight and specific responses at the transcription stage, the fission yeast fbp1 gene has been analyzed as a model system for decades. The fission yeast fbp1 gene is tightly repressed in the presence of glucose, and this gene is induced by over three orders of magnitude upon glucose starvation with a cascade of multi-layered regulations on various levels of genome and chromatin architecture. In this review article, we summarize the multi-layered transcriptional regulatory systems revealed by the analysis of the fission yeast fbp1 gene as a model system.

SPRTN and TDP1/TDP2 Independently Suppress 5-Aza-2'-deoxycytidine-Induced Genomic Instability in Human TK6 Cell Line.

DNA-protein cross-links (DPCs) are generated by internal factors such as cellular aldehydes that are generated during normal metabolism and external factors such as environmental mutagens. A nucleoside analog, 5-aza-2'-deoxycytidine (5-azadC), is randomly incorporated into the genome during DNA replication and binds DNA methyltransferase 1 (DNMT1) covalently to form DNMT1-DPCs without inducing DNA strand breaks. Despite the recent progress in understanding the mechanisms of DPCs repair, how DNMT1-DPCs are repaired is unclear. The metalloprotease SPRTN has been considered as the primary enzyme to degrade protein components of DPCs to initiate the repair of DPCs. In this study, we showed that SPRTN-deficient (SPRTN-/-) human TK6 cells displayed high sensitivity to 5-azadC, and the removal of 5-azadC-induced DNMT1-DPCs was significantly slower in SPRTN-/- cells than that in wild-type cells. We also showed that the ubiquitination-dependent proteasomal degradation, which was independent of the SPRTN-mediated processing, was also involved in the repair of DNMT1-DPCs. Unexpectedly, we found that cells that are double deficient in tyrosyl DNA phosphodiesterase 1 and 2 (TDP1-/-TDP2-/-) were also sensitive to 5-azadC, although the removal of 5-azadC-induced DNMT1-DPCs was not compromised significantly. Furthermore, the 5-azadC treatment induced a marked accumulation of chromosomal breaks in SPRTN-/- as well as TDP1-/-TDP2-/- cells compared to wild-type cells, strongly suggesting that the 5-azadC-induced cell death was attributed to chromosomal DNMT1-DPCs. We conclude that SPRTN protects cells from 5-azadC-induced DNMT1-DPCs, and SPRTN may play a direct proteolytic role against DNMT1-DPCs and TDP1/TDP2 also contributes to suppress genome instability caused by 5-azadC in TK6 cells.

A high-throughput 384-well CometChip platform reveals a role for 3-methyladenine in the cellular response to etoposide-induced DNA damage.

The Comet or single-cell gel electrophoresis assay is a highly sensitive method to measure cellular, nuclear genome damage. However, low throughput can limit its application for large-scale studies. To overcome these limitations, a 96-well CometChip platform was recently developed that increases throughput and reduces variation due to simultaneous processing and automated analysis of 96 samples. To advance throughput further, we developed a 384-well CometChip platform that allows analysis of ∼100 cells per well. The 384-well CometChip extends the capacity by 4-fold as compared to the 96-well system, enhancing application for larger DNA damage analysis studies. The overall sensitivity of the 384-well CometChip is consistent with that of the 96-well system, sensitive to genotoxin exposure and to loss of DNA repair capacity. We then applied the 384-well platform to screen a library of protein kinase inhibitors to probe each as enhancers of etoposide induced DNA damage. Here, we found that 3-methyladenine significantly increased levels of etoposide-induced DNA damage. Our results suggest that a 384-well CometChip is useful for large-scale DNA damage analyses, which may have increased potential in the evaluation of chemotherapy efficacy, compound library screens, population-based analyses of genome damage and evaluating the impact of environmental genotoxins on genome integrity.

XRCC1 counteracts poly(ADP ribose)polymerase (PARP) poisons, olaparib and talazoparib, and a clinical alkylating agent, temozolomide, by promoting the removal of trapped PARP1 from broken DNA.

Base excision repair (BER) removes damaged bases by generating single-strand breaks (SSBs), gap-filling by DNA polymerase ß (POLß), and resealing SSBs. A base-damaging agent, methyl methanesulfonate (MMS) is widely used to study BER. BER increases cellular tolerance to MMS, anti-cancer base-damaging drugs, temozolomide, carmustine, and lomustine, and to clinical poly(ADP ribose)polymerase (PARP) poisons, olaparib and talazoparib. The poisons stabilize PARP1/SSB complexes, inhibiting access of BER factors to SSBs. PARP1 and XRCC1 collaboratively promote SSB resealing by recruiting POLß to SSBs, but XRCC1-/- cells are much more sensitive to MMS than PARP1-/- cells. We recently report that the PARP1 loss in XRCC1-/- cells restores their MMS tolerance and conclude that XPCC1 facilitates the release of PARP1 from SSBs by maintaining its autoPARylation. We here show that the PARP1 loss in XRCC1-/- cells also restores their tolerance to the three anti-cancer base-damaging drugs, although they and MMS induce different sets of base damage. We reveal the synthetic lethality of the XRCC1-/- mutation, but not POLß-/- , with olaparib and talazoparib, indicating that XRCC1 is a unique BER factor in suppressing toxic PARP1/SSB complex and can suppress even when PARP1 catalysis is inhibited. In conclusion, XRCC1 suppresses the PARP1/SSB complex via PARP1 catalysis-dependent and independent mechanisms.

Construction of a human hTERT RPE-1 cell line with inducible Cre for editing of endogenous genes.

The human retinal pigment epithelial RPE-1 cell line immortalized with hTERT retains a stable karyotype with a modal chromosome number of 46 and has been widely used to study physiological events in human cell culture systems. To facilitate inducible knock-out or knock-in experiments in this cell line, we have modified the AAVS1 locus to harbour a DNA fragment encoding ERT2-Cre-ERT2 fusion protein under regulation of a Tet-On expression system. In the generated cell line, active Cre recombinase was induced by simple addition of doxycycline and tamoxifen to the culture medium. As proof of concept, we successfully introduced an oncogenic point mutation to the endogenous KRAS gene locus of this cell line. The cell line will serve as a powerful tool to conduct functional analyses of human genes.

Vertebrate CTF18 and DDX11 essential function in cohesion is bypassed by preventing WAPL-mediated cohesin release.

The alternative PCNA loader containing CTF18-DCC1-CTF8 facilitates sister chromatid cohesion (SCC) by poorly defined mechanisms. Here we found that in DT40 cells, CTF18 acts complementarily with the Warsaw breakage syndrome DDX11 helicase in mediating SCC and proliferation. We uncover that the lethality and cohesion defects of ctf18 ddx11 mutants are associated with reduced levels of chromatin-bound cohesin and rescued by depletion of WAPL, a cohesin-removal factor. On the contrary, high levels of ESCO1/2 acetyltransferases that acetylate cohesin to establish SCC do not rescue ctf18 ddx11 phenotypes. Notably, the tight proximity of sister centromeres and increased anaphase bridges characteristic of WAPL-depleted cells are abrogated by loss of both CTF18 and DDX11 The results reveal that vertebrate CTF18 and DDX11 collaborate to provide sufficient amounts of chromatin-loaded cohesin available for SCC generation in the presence of WAPL-mediated cohesin-unloading activity. This process modulates chromosome structure and is essential for cellular proliferation in vertebrates.

Reciprocal stabilization of transcription factor binding integrates two signaling pathways to regulate fission yeast fbp1 transcription.

Transcriptional regulation, a pivotal biological process by which cells adapt to environmental fluctuations, is achieved by the binding of transcription factors to target sequences in a sequence-specific manner. However, how transcription factors recognize the correct target from amongst the numerous candidates in a genome has not been fully elucidated. We here show that, in the fission-yeast fbp1 gene, when transcription factors bind to target sequences in close proximity, their binding is reciprocally stabilized, thereby integrating distinct signal transduction pathways. The fbp1 gene is massively induced upon glucose starvation by the activation of two transcription factors, Atf1 and Rst2, mediated via distinct signal transduction pathways. Atf1 and Rst2 bind to the upstream-activating sequence 1 region, carrying two binding sites located 45 bp apart. Their binding is reciprocally stabilized due to the close proximity of the two target sites, which destabilizes the independent binding of Atf1 or Rst2. Tup11/12 (Tup-family co-repressors) suppress independent binding. These data demonstrate a previously unappreciated mechanism by which two transcription-factor binding sites, in close proximity, integrate two independent-signal pathways, thereby behaving as a hub for signal integration.

Targeting chromosome trisomy for chromosome editing.

A trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection markers. Upon construction of cells carrying markers targeted in one of the trisomic chromosome 2s, cells that have lost markers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes.

Follow-up genotoxicity assessment of Ames-positive/equivocal chemicals using the improved thymidine kinase gene mutation assay in DNA repair-deficient human TK6 cells.

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to develop in vitro genotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoid TK6 cells lacking XRCC1 and XPA (XRCC1-/-/XPA-/-), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficient TK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA), p-phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment using XRCC1-/-/XPA-/- cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-up in vivo studies. In contrast, the mutant frequency was markedly increased in XRCC1-/-/XPA-/- cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances.

Division of labor of Y-family polymerases in translesion-DNA synthesis for distinct types of DNA damage.

Living organisms are continuously under threat from a vast array of DNA-damaging agents, which impact genome DNA. DNA replication machinery stalls at damaged template DNA. The stalled replication fork is restarted via bypass replication by translesion DNA-synthesis polymerases, including the Y-family polymerases Polη, Polι, and Polκ, which possess the ability to incorporate nucleotides opposite the damaged template. To investigate the division of labor among these polymerases in vivo, we generated POLη-/-, POLι-/-, POLκ-/-, double knockout (KO), and triple knockout (TKO) mutants in all combinations from human TK6 cells. TKO cells exhibited a hypersensitivity to ultraviolet (UV), cisplatin (CDDP), and methyl methanesulfonate (MMS), confirming the pivotal role played by these polymerases in bypass replication of damaged template DNA. POLη-/- cells, but not POLι-/- or POLκ-/- cells, showed a strong sensitivity to UV and CDDP, while TKO cells showed a slightly higher sensitivity to UV and CDDP than did POLη-/- cells. On the other hand, TKO cells, but not all single KO cells, exhibited a significantly higher sensitivity to MMS than did wild-type cells. Consistently, DNA-fiber assay revealed that Polη plays a crucial role in bypassing lesions caused by UV-mimetic agent 4-nitroquinoline-1-oxide and CDDP, while all three polymerases play complementary roles in bypassing MMS-induced damage. Our findings indicate that the three Y-family polymerases play distinctly different roles in bypass replication, according to the type of DNA damage generated on the template strand.