Publications by authors named "Hiroyuki Sasanuma"

52 Publications

Protein phosphatase 1 acts as a RIF1 effector to suppress DSB resection prior to Shieldin action.

Cell Rep 2021 Jul;36(2):109383

Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-Cho, Toyonaka, Osaka 560-0043, Japan. Electronic address:

DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining (NHEJ) or homologous recombination (HR). RIF1 negatively regulates resection through the effector Shieldin, which associates with a short 3' single-stranded DNA (ssDNA) overhang by the MRN (MRE11-RAD50-NBS1) complex, to prevent further resection and HR repair. In this study, we show that RIF1, but not Shieldin, inhibits the accumulation of CtIP at DSB sites immediately after damage, suggesting that RIF1 has another effector besides Shieldin. We find that protein phosphatase 1 (PP1), a known RIF1 effector in replication, localizes at damage sites dependent on RIF1, where it suppresses downstream CtIP accumulation and limits the resection by the MRN complex. PP1 therefore acts as a RIF1 effector distinct from Shieldin. Furthermore, PP1 deficiency in the context of Shieldin depletion elevates HR immediately after irradiation. We conclude that PP1 inhibits resection before the action of Shieldin to prevent precocious HR in the early phase of the damage response.
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http://dx.doi.org/10.1016/j.celrep.2021.109383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8293623PMC
July 2021

FANCD2-associated nuclease 1 partially compensates for the lack of Exonuclease 1 in mismatch repair.

Mol Cell Biol 2021 Jul 6:MCB0030321. Epub 2021 Jul 6.

Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

Germline mutations in the mismatch repair (R) genes , , and are linked to cancer of the colon and other organs, characterised by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in , encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1. Analysis of the MLH1 interactome identified FANCD2-associated nuclease 1 (FAN1), a novel enzyme with biochemical properties resembling EXO1. We now show that FAN1 efficiently substitutes for EXO1 in MMR assays and that this functional complementation is modulated by its interaction with MLH1. FAN1 also contributes towards MMR : cells lacking both EXO1 and FAN1 have a MMR defect and display resistance to -methyl--nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, implying that the two nucleases act redundantly in the same antimutagenic pathway. However, the increased drug resistance and mutator phenotype of FAN1/EXO1-deficient cells are less prominent than those seen in cells lacking MSH6 or MLH1. Eukaryotic cells thus apparently possess additional mechanisms that compensate for the loss of EXO1.
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http://dx.doi.org/10.1128/MCB.00303-21DOI Listing
July 2021

Fanconi anemia proteins participate in a break-induced-replication-like pathway to counter replication stress.

Nat Struct Mol Biol 2021 Jun 10;28(6):487-500. Epub 2021 Jun 10.

State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.

Fanconi anemia (FA) is a devastating hereditary disease characterized by bone marrow failure (BMF) and acute myeloid leukemia (AML). As FA-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), ICLs are widely assumed to be the lesions responsible for FA symptoms. Here, we show that FA-mutated cells are hypersensitive to persistent replication stress and that FA proteins play a role in the break-induced-replication (BIR)-like pathway for fork restart. Both the BIR-like pathway and ICL repair share almost identical molecular mechanisms of 53BP1-BRCA1-controlled signaling response, SLX4- and FAN1-mediated fork cleavage and POLD3-dependent DNA synthesis, suggesting that the FA pathway is intrinsically one of the BIR-like pathways. Replication stress not only triggers BMF in FA-deficient mice, but also specifically induces monosomy 7, which is associated with progression to AML in patients with FA, in FA-deficient cells.
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http://dx.doi.org/10.1038/s41594-021-00602-9DOI Listing
June 2021

XRCC1 prevents toxic PARP1 trapping during DNA base excision repair.

Mol Cell 2021 07 7;81(14):3018-3030.e5. Epub 2021 Jun 7.

Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, UK; Department of Genome Dynamics, Institute of Molecular Genetics of the Czech Academy of Sciences, 142 20 Prague 4, Czech Republic. Electronic address:

Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1 cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.
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http://dx.doi.org/10.1016/j.molcel.2021.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294329PMC
July 2021

Epigenetic suppression of SLFN11 in germinal center B-cells during B-cell development.

PLoS One 2021 29;16(1):e0237554. Epub 2021 Jan 29.

Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.

Background: SLFN11 has recently been reported to execute cancer cells harboring replicative stress induced by DNA damaging agents. However, the roles of SLFN11 under physiological conditions remain poorly understood. Germinal center B-cells (GCBs) undergo somatic hypermutations and class-switch recombination, which can cause physiological genotoxic stress. Hence, we tested whether SLFN11 expression needs to be suppressed in GCBs during B-cell development.

Objective: To clarify the expression profile of SLFN11 in different developmental stages of B-cells and B-cell-derived cancers.

Methods: We analyzed the expression of SLFN11 by mining cell line databases for different stages of normal B-cells and various types of B-cell-derived cancer cell lines. We performed dual immunohistochemical staining for SLFN11 and B-cell specific markers in normal human lymphatic tissues. We tested the effects of two epigenetic modifiers, an EZH2 inhibitor, tazemetostat (EPZ6438) and a histone deacetylase inhibitor, panobinostat (LBH589) on SLFN11 expression in GCB-derived lymphoma cell lines. We also examined the therapeutic efficacy of these drugs in combination with cytosine arabinoside and the effects of SLFN11 on the efficacy of cytosine arabinoside in SLFN11-overexpressing cells.

Results: SLFN11 mRNA level was found low in both normal GCBs and GCB-DLBCL (GCB like-diffuse large B-cell lymphoma). Immunohistochemical staining showed low SLFN11 expression in GCBs and high SLFN11 expression in plasmablasts and plasmacytes. The EZH2 and HDAC epigenetic modifiers upregulated SLFN11 expression in GCB-derived lymphoma cells and made them more susceptible to cytosine arabinoside. SLFN11 overexpression further sensitized GCB-derived lymphoma cells to cytosine arabinoside.

Conclusions: The expression of SLFN11 is epigenetically suppressed in normal GCBs and GCB-derived lymphomas. GCB-derived lymphomas with low SLFN11 expression can be treated by the combination of epigenetic modifiers and cytosine arabinoside.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237554PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846023PMC
April 2021

Genetic evidence for the involvement of mismatch repair proteins, PMS2 and MLH3, in a late step of homologous recombination.

J Biol Chem 2020 12;295(51):17460-17475

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan. Electronic address:

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3 and PMS2 mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3 and PMS2 cells. MLH3 and MLH3 cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme-induced double-strand breaks. PMS2 and PMS2 cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3 and PMS2 mutations had an additive effect on the heteroallelic HR. MLH3/PMS2 cells showed normal kinetics of γ-irradiation-induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3/PMS2 cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.
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http://dx.doi.org/10.1074/jbc.RA120.013521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762965PMC
December 2020

Tyrosyl-DNA phosphodiesterases are involved in mutagenic events at a ribonucleotide embedded into DNA in human cells.

PLoS One 2020 31;15(12):e0244790. Epub 2020 Dec 31.

Department of Biology, Graduate School of Science, Chiba University, Chiba, Japan.

Ribonucleoside triphosphates are often incorporated into genomic DNA during DNA replication. The accumulation of unrepaired ribonucleotides is associated with genomic instability, which is mediated by DNA topoisomerase 1 (Top1) processing of embedded ribonucleotides. The cleavage initiated by Top1 at the site of a ribonucleotide leads to the formation of a Top1-DNA cleavage complex (Top1cc), occasionally resulting in a DNA double-strand break (DSB). In humans, tyrosyl-DNA phosphodiesterases (TDPs) are essential repair enzymes that resolve the trapped Top1cc followed by downstream repair factors. However, there is limited cellular evidence of the involvement of TDPs in the processing of incorporated ribonucleotides in mammals. We assessed the role of TDPs in mutagenesis induced by a single ribonucleotide embedded into DNA. A supF shuttle vector site-specifically containing a single riboguanosine (rG) was introduced into the human lymphoblastoid TK6 cell line and its TDP1-, TDP2-, and TDP1/TDP2-deficient derivatives. TDP1 and TDP2 insufficiency remarkably decreased the mutant frequency caused by an embedded rG. The ratio of large deletion mutations induced by rG was also substantially lower in TDP1/TDP2-deficient cells than wild-type cells. Furthermore, the disruption of TDPs reduced the length of rG-mediated large deletion mutations. The recovery ratio of the propagated plasmid was also increased in TDP1/TDP2-deficient cells after the transfection of the shuttle vector containing rG. The results suggest that TDPs-mediated ribonucleotide processing cascade leads to unfavorable consequences, whereas in the absence of these repair factors, a more error-free processing pathway might function to suppress the ribonucleotide-induced mutagenesis. Furthermore, base substitution mutations at sites outside the position of rG were detected in the supF gene via a TDPs-independent mechanism. Overall, we provide new insights into the mechanism of mutagenesis induced by an embedded ribonucleotide in mammalian cells, which may lead to the fatal phenotype in the ribonucleotide excision repair deficiency.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0244790PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775084PMC
March 2021

Restoration of ligatable "clean" double-strand break ends is the rate-limiting step in the rejoining of ionizing-radiation-induced DNA breakage.

DNA Repair (Amst) 2020 09;93:102913

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto, 606-8501, Japan. Electronic address:

Radiotherapy kills malignant cells by generating double-strand breaks (DSBs). Ionizing- radiation (IR) generates "dirty" DSBs, which associates with blocking chemical adducts at DSB ends. Homologous-directed repair (HDR) efficiently removes IR-induced blocking adducts from both 3' and 5' ends of DSBs. Nonhomologous end-joining (NHEJ) rejoins virtually all DSBs in G phase and ∼80 % of DSBs in G phase. However, DNA Ligase IV, an essential NHEJ factor, rejoins only "clean" ligatable DSBs carrying 3'-OH and 5'-phosphate DSB ends but not dirty DSBs. Recent studies have identified a number of nucleases, especially the MRE11 nuclease, as key factors performing the removal of blocking chemical adducts to restore clean ligatable DSBs for subsequent NHEJ. This restoration, but not subsequent NHEJ, is the rate-limiting step in the rejoining of IR- induced DSBs. This review describes repair factors that contribute to the restoration of clean DSBs before NHEJ.
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http://dx.doi.org/10.1016/j.dnarep.2020.102913DOI Listing
September 2020

Participation of TDP1 in the repair of formaldehyde-induced DNA-protein cross-links in chicken DT40 cells.

PLoS One 2020 26;15(6):e0234859. Epub 2020 Jun 26.

Division of Radiation Life Science, Institute for Integrated Radiation and Nuclear Science, Kyoto University, Kumatori, Osaka, Japan.

Proteins are covalently trapped on DNA to form DNA-protein cross-links (DPCs) when cells are exposed to DNA-damaging agents. Aldehyde compounds produce common types of DPCs that contain proteins in an undisrupted DNA strand. Tyrosyl-DNA phosphodiesterase 1 (TDP1) repairs topoisomerase 1 (TOPO1) that is trapped at the 3'-end of DNA. In the present study, we examined the contribution of TDP1 to the repair of formaldehyde-induced DPCs using a reverse genetic strategy with chicken DT40 cells. The results obtained showed that cells deficient in TDP1 were sensitive to formaldehyde. The removal of formaldehyde-induced DPCs was slower in tdp1-deficient cells than in wild type cells. We also found that formaldehyde did not produce trapped TOPO1, indicating that trapped TOPO1 was not a primary cytotoxic DNA lesion that was generated by formaldehyde and repaired by TDP1. The formaldehyde treatment resulted in the accumulation of chromosomal breakages that were more prominent in tdp1-deficient cells than in wild type cells. Therefore, TDP1 plays a critical role in the repair of formaldehyde-induced DPCs that are distinct from trapped TOPO1.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234859PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319324PMC
September 2020

Topoisomerase I-driven repair of UV-induced damage in NER-deficient cells.

Proc Natl Acad Sci U S A 2020 06 8;117(25):14412-14420. Epub 2020 Jun 8.

Department of Radiation Genetics, Kyoto University, Graduate School of Medicine, 606-8501 Kyoto, Japan;

Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1.
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http://dx.doi.org/10.1073/pnas.1920165117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321995PMC
June 2020

UBC13-Mediated Ubiquitin Signaling Promotes Removal of Blocking Adducts from DNA Double-Strand Breaks.

iScience 2020 Apr 31;23(4):101027. Epub 2020 Mar 31.

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address:

Chemical modifications and adducts at DNA double-strand break (DSB) ends must be cleaned before re-joining by non-homologous end-joining (NHEJ). MRE11 nuclease is essential for efficient removal of Topoisomerase II (TOP2)-DNA adducts from TOP2 poison-induced DSBs. However, mechanisms in MRE11 recruitment to DSB sites in G phase remain poorly understood. Here, we report that TOP2-DNA adducts are expeditiously removed through UBC13-mediated polyubiquitination, which promotes DSB resection in G phase. We found that this ubiquitin signaling is required for efficient recruitment of MRE11 onto DSB sites in G by facilitating localization of RAP80 and BRCA1 to DSB sites and complex formation between BRCA1 and MRE11 at DSB sites. UBC13 and MRE11 are dispensable for restriction-enzyme-induced "clean" DSBs repair but responsible for over 50% and 70% of NHEJ-dependent repair of γ-ray-induced "dirty" DSBs, respectively. In conclusion, ubiquitin signaling promotes nucleolytic removal of DSB blocking adducts by MRE11 before NHEJ.
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http://dx.doi.org/10.1016/j.isci.2020.101027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155233PMC
April 2020

TDP2 suppresses genomic instability induced by androgens in the epithelial cells of prostate glands.

Genes Cells 2020 Jul 5;25(7):450-465. Epub 2020 May 5.

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Androgens stimulate the proliferation of epithelial cells in the prostate by activating topoisomerase 2 (TOP2) and regulating the transcription of target genes. TOP2 resolves the entanglement of genomic DNA by transiently generating double-strand breaks (DSBs), where TOP2 homodimers covalently bind to 5' DSB ends, called TOP2-DNA cleavage complexes (TOP2ccs). When TOP2 fails to rejoin TOP2ccs generating stalled TOP2ccs, tyrosyl DNA phosphodiesterase-2 (TDP2) removes 5' TOP2 adducts from stalled TOP2ccs prior to the ligation of the DSBs by nonhomologous end joining (NHEJ), the dominant DSB repair pathway in G /G phases. We previously showed that estrogens frequently generate stalled TOP2ccs in G /G phases. Here, we show that physiological concentrations of androgens induce several DSBs in individual human prostate cancer cells during G phase, and loss of TDP2 causes a five times higher number of androgen-induced chromosome breaks in mitotic chromosome spreads. Intraperitoneally injected androgens induce several DSBs in individual epithelial cells of the prostate in TDP2-deficient mice, even at 20 hr postinjection. In conclusion, physiological concentrations of androgens have very strong genotoxicity, most likely by generating stalled TOP2ccs.
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http://dx.doi.org/10.1111/gtc.12770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497232PMC
July 2020

Enhancing the sensitivity of the thymidine kinase assay by using DNA repair-deficient human TK6 cells.

Environ Mol Mutagen 2020 07 15;61(6):602-610. Epub 2020 Apr 15.

Department of Radiation Genetics, Kyoto University, Graduate School of Medicine, Kyoto, Japan.

The OECD guidelines define the bioassays of identifying mutagenic chemicals, including the thymidine kinase (TK) assay, which specifically detects the mutations that inactivate the TK gene in the human TK6 lymphoid line. However, the sensitivity of this assay is limited because it detects mutations occurring only in the TK gene but not any other genes. Moreover, the limited sensitivity of the conventional TK assay is caused by the usage of DNA repair-proficient wild-type cells, which are capable of accurately repairing DNA damage induced by chemicals. Mutagenic chemicals produce a variety of DNA lesions, including base lesions, sugar damage, crosslinks, and strand breaks. Base damage causes point mutations and is repaired by the base excision repair (BER) and nucleotide excision repair (NER) pathways. To increase the sensitivity of TK assay, we simultaneously disrupted two genes encoding XRCC1, an important BER factor, and XPA, which is essential for NER, generating XRCC1 /XPA cells from TK6 cells. We measured the mutation frequency induced by four typical mutagenic agents, methyl methane sulfonate (MMS), cis-diamminedichloro-platinum(II) (cisplatin, CDDP), mitomycin-C (MMC), and cyclophosphamide (CP) by the conventional TK assay using wild-type TK6 cells and also by the TK assay using XRCC1 /XPA cells. The usage of XRCC1 /XPA cells increased the sensitivity of detecting the mutagenicity by 8.6 times for MMC, 8.5 times for CDDP, and 2.6 times for MMS in comparison with the conventional TK assay. In conclusion, the usage of XRCC1 /XPA cells will significantly improve TK assay.
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http://dx.doi.org/10.1002/em.22371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384079PMC
July 2020

Estrogen Induces Mammary Ductal Dysplasia via the Upregulation of Myc Expression in a DNA-Repair-Deficient Condition.

iScience 2020 Feb 9;23(2):100821. Epub 2020 Jan 9.

Department of Breast Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.

Mammary ductal dysplasia is a phenotype observed in precancerous lesions and early-stage breast cancer. However, the mechanism of dysplasia formation remains elusive. Here we show, by establishing a novel dysplasia model system, that estrogen, a female hormone, has the potential to cause mammary ductal dysplasia. We injected estradiol (E2), the most active form of estrogen, daily into scid mice with a defect in non-homologous end joining repair and observed dysplasia formation with cell proliferation at day 30. The protooncogene Myc is a downstream target of estrogen signaling, and we found that its expression is augmented in mammary epithelial cells in this dysplasia model. Treatment with a Myc inhibitor reduced E2-induced dysplasia formation. Moreover, we found that isoflavones inhibited E2-induced dysplasia formation. Our dysplasia model system provides insights into the mechanistic understanding of breast tumorigenesis and the development of breast cancer prevention.
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http://dx.doi.org/10.1016/j.isci.2020.100821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976935PMC
February 2020

Type II DNA Topoisomerases Cause Spontaneous Double-Strand Breaks in Genomic DNA.

Genes (Basel) 2019 10 30;10(11). Epub 2019 Oct 30.

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606-8501, Japan.

Type II DNA topoisomerase enzymes (TOP2) catalyze topological changes by strand passage reactions. They involve passing one intact double stranded DNA duplex through a transient enzyme-bridged break in another (gated helix) followed by ligation of the break by TOP2. A TOP2 poison, etoposide blocks TOP2 catalysis at the ligation step of the enzyme-bridged break, increasing the number of stable TOP2 cleavage complexes (TOP2ccs). Remarkably, such pathological TOP2ccs are formed during the normal cell cycle as well as in postmitotic cells. Thus, this 'abortive catalysis' can be a major source of spontaneously arising DNA double-strand breaks (DSBs). TOP2-mediated DSBs are also formed upon stimulation with physiological concentrations of androgens and estrogens. The frequent occurrence of TOP2-mediated DSBs was previously not appreciated because they are efficiently repaired. This repair is performed in collaboration with BRCA1, BRCA2, MRE11 nuclease, and tyrosyl-DNA phosphodiesterase 2 (TDP2) with nonhomologous end joining (NHEJ) factors. This review first discusses spontaneously arising DSBs caused by the abortive catalysis of TOP2 and then summarizes proteins involved in repairing stalled TOP2ccs and discusses the genotoxicity of the sex hormones.
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http://dx.doi.org/10.3390/genes10110868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895833PMC
October 2019

Processing of a single ribonucleotide embedded into DNA by human nucleotide excision repair and DNA polymerase η.

Sci Rep 2019 09 26;9(1):13910. Epub 2019 Sep 26.

Department of Biology, Graduate School of Science, Chiba University, Chiba, 263-8522, Japan.

DNA polymerases often incorporate non-canonical nucleotide, i.e., ribonucleoside triphosphates into the genomic DNA. Aberrant accumulation of ribonucleotides in the genome causes various cellular abnormalities. Here, we show the possible role of human nucleotide excision repair (NER) and DNA polymerase η (Pol η) in processing of a single ribonucleotide embedded into DNA. We found that the reconstituted NER system can excise the oxidized ribonucleotide on the plasmid DNA. Taken together with the evidence that Pol η accurately bypasses a ribonucleotide, i.e., riboguanosine (rG) or its oxidized derivative (8-oxo-rG) in vitro, we further assessed the mutagenic potential of the embedded ribonucleotide in human cells lacking NER or Pol η. A single rG on the supF reporter gene predominantly induced large deletion mutations. An embedded 8-oxo-rG caused base substitution mutations at the 3'-neighboring base rather than large deletions in wild-type cells. The disruption of XPA, an essential factor for NER, or Pol η leads to the increased mutant frequency of 8-oxo-rG. Furthermore, the frequency of 8-oxo-rG-mediated large deletions was increased by the loss of Pol η, but not XPA. Collectively, our results suggest that base oxidation of the embedded ribonucleotide enables processing of the ribonucleotide via alternative DNA repair and damage tolerance pathways.
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http://dx.doi.org/10.1038/s41598-019-50421-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763444PMC
September 2019

Spatial Chromosome Folding and Active Transcription Drive DNA Fragility and Formation of Oncogenic MLL Translocations.

Mol Cell 2019 07 12;75(2):267-283.e12. Epub 2019 Jun 12.

Institute of Molecular Biology (IMB), Mainz 55128, Germany. Electronic address:

How spatial chromosome organization influences genome integrity is still poorly understood. Here, we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CCCTC-binding factor (CTCF) and cohesin-bound sites at the bases of chromatin loops, and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hotspots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability and are key contributors to the occurrence of genome rearrangements that drive cancer.
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http://dx.doi.org/10.1016/j.molcel.2019.05.015DOI Listing
July 2019

Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis.

Chromosoma 2019 09 6;128(3):453-471. Epub 2019 Jun 6.

Institute for Protein Research, Graduate School of Science, Osaka University, Suita, Osaka, Japan.

Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei, but not that of a meiosis-specific Dmc1. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and/or multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphases I and II, suggesting the absence of DNA damage-induced cell cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase I.
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http://dx.doi.org/10.1007/s00412-019-00709-5DOI Listing
September 2019

PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching.

PLoS One 2019 6;14(3):e0213383. Epub 2019 Mar 6.

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0213383PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402704PMC
December 2019

BRCA1 Haploinsufficiency Is Masked by RNF168-Mediated Chromatin Ubiquitylation.

Mol Cell 2019 03 28;73(6):1267-1281.e7. Epub 2019 Jan 28.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA. Electronic address:

BRCA1 functions at two distinct steps during homologous recombination (HR). Initially, it promotes DNA end resection, and subsequently it recruits the PALB2 and BRCA2 mediator complex, which stabilizes RAD51-DNA nucleoprotein filaments. Loss of 53BP1 rescues the HR defect in BRCA1-deficient cells by increasing resection, suggesting that BRCA1's downstream role in RAD51 loading is dispensable when 53BP1 is absent. Here we show that the E3 ubiquitin ligase RNF168, in addition to its canonical role in inhibiting end resection, acts in a redundant manner with BRCA1 to load PALB2 onto damaged DNA. Loss of RNF168 negates the synthetic rescue of BRCA1 deficiency by 53BP1 deletion, and it predisposes BRCA1 heterozygous mice to cancer. BRCA1RNF168 cells lack RAD51 foci and are hypersensitive to PARP inhibitor, whereas forced targeting of PALB2 to DNA breaks in mutant cells circumvents BRCA1 haploinsufficiency. Inhibiting the chromatin ubiquitin pathway may, therefore, be a synthetic lethality strategy for BRCA1-deficient cancers.
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http://dx.doi.org/10.1016/j.molcel.2018.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430682PMC
March 2019

SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines.

Proc Natl Acad Sci U S A 2018 12 28;115(50):12793-12798. Epub 2018 Nov 28.

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan

DNA damage tolerance (DDT) releases replication blockage caused by damaged nucleotides on template strands employing two alternative pathways, error-prone translesion DNA synthesis (TLS) and error-free template switch (TS). Lys164 of proliferating cell nuclear antigen (PCNA) is SUMOylated during the physiological cell cycle. To explore the role for SUMOylation of PCNA in DDT, we characterized chicken DT40 and human TK6 B cells deficient in the PIAS1 and PIAS4 small ubiquitin-like modifier (SUMO) E3 ligases. DT40 cells have a unique advantage in the phenotypic analysis of DDT as they continuously diversify their immunoglobulin (Ig) variable genes by TLS and TS [Ig gene conversion (GC)], both relieving replication blocks at abasic sites without accompanying by DNA breakage. Remarkably, cells displayed a multifold decrease in SUMOylation of PCNA at Lys164 and over a 90% decrease in the rate of TS. Likewise, TK6 cells showed a shift of DDT from TS to TLS at a chemosynthetic UV lesion inserted into the genomic DNA. The mutation caused a ∼90% decrease in the rate of Ig GC and no additional impact on cells. This epistatic relationship between the and the mutations suggests that PIAS1 and PIAS4 promote TS mainly through SUMOylation of PCNA at Lys164. This idea is further supported by the data that overexpression of a PCNA-SUMO1 chimeric protein restores defects in TS in cells. In conclusion, SUMOylation of PCNA at Lys164 promoted by PIAS1 and PIAS4 ensures the error-free release of replication blockage during physiological DNA replication in metazoan cells.
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http://dx.doi.org/10.1073/pnas.1716349115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294928PMC
December 2018

BRCA1 ensures genome integrity by eliminating estrogen-induced pathological topoisomerase II-DNA complexes.

Proc Natl Acad Sci U S A 2018 11 23;115(45):E10642-E10651. Epub 2018 Oct 23.

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan;

Women having BRCA1 germ-line mutations develop cancer in breast and ovary, estrogen-regulated tissues, with high penetrance. Binding of estrogens to the estrogen receptor (ER) transiently induces DNA double-strand breaks (DSBs) by topoisomerase II (TOP2) and controls gene transcription. TOP2 resolves catenated DNA by transiently generating DSBs, TOP2-cleavage complexes (TOP2ccs), where TOP2 covalently binds to 5' ends of DSBs. TOP2 frequently fails to complete its catalysis, leading to formation of pathological TOP2ccs. We have previously shown that the endonucleolytic activity of MRE11 plays a key role in removing 5' TOP2 adducts in G phase. We show here that BRCA1 promotes MRE11-mediated removal of TOP2 adducts in G phase. We disrupted the gene in -deficient ER-positive breast cancer and B cells. The loss of BRCA1 caused marked increases of pathological TOP2ccs in G phase following exposure to etoposide, which generates pathological TOP2ccs. We conclude that BRCA1 promotes the removal of TOP2 adducts from DSB ends for subsequent nonhomologous end joining. -deficient cells showed a decrease in etoposide-induced MRE11 foci in G phase, suggesting that BRCA1 repairs pathological TOP2ccs by promoting the recruitment of MRE11 to TOP2cc sites. BRCA1 depletion also leads to the increase of unrepaired DSBs upon estrogen treatment both in vitro in G-arrested breast cancer cells and in vivo in epithelial cells of mouse mammary glands. BRCA1 thus plays a critical role in removing pathological TOP2ccs induced by estrogens as well as etoposide. We propose that BRCA1 suppresses tumorigenesis by removing estrogen-induced pathological TOP2ccs throughout the cell cycle.
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http://dx.doi.org/10.1073/pnas.1803177115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233096PMC
November 2018

Differential micronucleus frequency in isogenic human cells deficient in DNA repair pathways is a valuable indicator for evaluating genotoxic agents and their genotoxic mechanisms.

Environ Mol Mutagen 2018 07 15;59(6):529-538. Epub 2018 May 15.

Department of Radiation Genetics, Kyoto University, Graduate School of Medicine, Yoshida Konoe, Sakyo-ku, Kyoto, 606-8501, Japan.

The micronucleus (MN) test has become an attractive tool both for evaluating the genotoxicity of test chemicals because of its ability to detect clastogenic and aneugenic events and for its convenience. As the MN assay has been mostly performed using only DNA repair-proficient mammalian cells, we believed that the comparison of the MN frequency between DNA repair-proficient and -deficient human cells may be an excellent indicator for detecting the genotoxic potential of test chemicals and for understanding their mode of action. To address this issue, the following five genes encoding DNA-damage-response (DDR) factors were disrupted in the TK6 B cell line, a human cell line widely used for the MN test: FANCD2, DNA polymerase ζ (REV3), XRCC1, RAD54, and/or LIG4. Using these isogenic TK6 cell lines, the MN test was conducted for four widely-used DNA-damaging agents: methyl methanesulfonate (MMS), hydrogen peroxide (H O ), γ-rays, and mitomycin C (MMC). The frequency of micronuclei in the double strand break repair-deficient RAD54 /LIG4 cells after exposure to γ-rays, H O , MMS and MMC was 6.2-7.5 times higher than that of parental wild-type TK6 cells. The percentages of cells exhibiting micronuclei in the base excision repair- and single strand break repair-deficient XRCC1 cells after exposure to H O , MMC and MMS were all ∼5 times higher than those of wild-type cells. In summary, a supplementary MN assay using the combination of RAD54 /LIG4 , XRCC1 and wild-type TK6 cells is a promising method for detecting the genotoxic potential of test chemicals and their mode of action. Environ. Mol. Mutagen., 2018. © 2018 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/em.22201DOI Listing
July 2018

Disruption of Hif-1α enhances cytotoxic effects of metformin in murine squamous cell carcinoma.

Int J Radiat Biol 2018 01 12;94(1):88-96. Epub 2017 Dec 12.

a Particle Radiation Biology, Division of Radiation Life Science , Research Reactor Institute, Kyoto University , Kumatori-cho, Osaka , Japan.

Purpose: In the present study, we investigated whether the disruption of the Hif-1α gene affects the sensitivity of SCC VII cells to metformin and also if metformin functions as a radiosensitizer using murine squamous cell carcinoma (SCC VII) cells.

Materials And Methods: Cultured SCC VII and SCC VII Hif-1α-deficient cells were incubated with metformin under glucose-free and/or hypoxia-mimetic conditions and cell viabilities were measured. Tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating cells. Tumor-bearing mice were then subjected to γ-ray irradiation after the metformin treatment. Immediately after irradiation, cells were isolated from some tumors and incubated with a cytokinesis blocker. The responses of quiescent and total (= proliferating + quiescent) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU.

Results: The disruption of Hif-1α increased the sensitivity of SCC VII cells to metformin in glucose-free medium. Metformin-induced decreases in the percentage of dead cells in the presence of CoCl were partially reduced when Hif-1α was disrupted. In vivo, metformin increased the radiosensitivity of SCC VII Hif-1α-deficient cells.

Conclusion: The combination of disruption of Hif-1α and metformin effectively enhanced the radiosensitivity of SCC VII cells.
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http://dx.doi.org/10.1080/09553002.2018.1409443DOI Listing
January 2018

Complementation of aprataxin deficiency by base excision repair enzymes in mitochondrial extracts.

Nucleic Acids Res 2017 Sep;45(17):10079-10088

Genome Integrity and Structural Biology Laboratory, DNA Repair and Nucleic Acid Enzymology Group, National Institutes of Health, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.

Mitochondrial aprataxin (APTX) protects the mitochondrial genome from the consequence of ligase failure by removing the abortive ligation product, i.e. the 5'-adenylate (5'-AMP) group, during DNA replication and repair. In the absence of APTX activity, blocked base excision repair (BER) intermediates containing the 5'-AMP or 5'-adenylated-deoxyribose phosphate (5'-AMP-dRP) lesions may accumulate. In the current study, we examined DNA polymerase (pol) γ and pol β as possible complementing enzymes in the case of APTX deficiency. The activities of pol β lyase and FEN1 nucleotide excision were able to remove the 5'-AMP-dRP group in mitochondrial extracts from APTX-/- cells. However, the lyase activity of purified pol γ was weak against the 5'-AMP-dRP block in a model BER substrate, and this activity was not able to complement APTX deficiency in mitochondrial extracts from APTX-/-Pol β-/- cells. FEN1 also failed to provide excision of the 5'-adenylated BER intermediate in mitochondrial extracts. These results illustrate the potential role of pol β in complementing APTX deficiency in mitochondria.
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http://dx.doi.org/10.1093/nar/gkx654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622373PMC
September 2017

Selective cytotoxicity of the anti-diabetic drug, metformin, in glucose-deprived chicken DT40 cells.

PLoS One 2017 19;12(9):e0185141. Epub 2017 Sep 19.

Division of Radiation Life Science, Research Reactor Institute, Kyoto University, Kumatori, Osaka, Japan.

Metformin is a biguanide drug that is widely used in the treatment of diabetes. Epidemiological studies have indicated that metformin exhibits anti-cancer activity. However, the molecular mechanisms underlying this activity currently remain unclear. We hypothesized that metformin is cytotoxic in a tumor-specific environment such as glucose deprivation and/or low oxygen (O2) tension. We herein demonstrated that metformin was highly cytotoxic under glucose-depleted, but not hypoxic (2% O2) conditions. In order to elucidate the underlying mechanisms of this selective cytotoxicity, we treated exposed DNA repair-deficient chicken DT40 cells with metformin under glucose-depleted conditions and measured cellular sensitivity. Under glucose-depleted conditions, metformin specifically killed fancc and fancl cells that were deficient in FANCC and FANCL proteins, respectively, which are involved in DNA interstrand cross-link repair. An analysis of chromosomal aberrations in mitotic chromosome spreads revealed that a clinically relevant concentration of metformin induced DNA double-strand breaks (DSBs) in fancc and fancl cells under glucose-depleted conditions. In summary, metformin induced DNA damage under glucose-depleted conditions and selectively killed cells. This metformin-mediated selective toxicity may suppress the growth of malignant tumors that are intrinsically deprived of glucose.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185141PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605006PMC
October 2017

TDP1 is Critical for the Repair of DNA Breaks Induced by Sapacitabine, a Nucleoside also Targeting ATM- and BRCA-Deficient Tumors.

Mol Cancer Ther 2017 11 11;16(11):2543-2551. Epub 2017 Aug 11.

Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, NIH, Bethesda, Maryland.

2'--cyano-2'-deoxy-1-β-d-arabino-pentofuranosylcytosine (CNDAC) is the active metabolite of the anticancer drug, sapacitabine. CNDAC is incorporated into the genome during DNA replication and subsequently undergoes β-elimination that generates single-strand breaks with abnormal 3'-ends. Because tyrosyl-DNA phosphodiesterase 1 (TDP1) selectively hydrolyzes nonphosphorylated 3'-blocking ends, we tested its role in the repair of CNDAC-induced DNA damage. We show that cells lacking TDP1 (avian DT40 cells and human TSCER2 and HCT116 cells) exhibit marked hypersensitivity to CNDAC. We also identified BRCA1, FANCD2, and PCNA in the DNA repair pathways to CNDAC. Comparing CNDAC with the chemically related arabinosyl nucleoside analog, cytosine arabinoside (cytarabine, AraC) and the topoisomerase I inhibitor camptothecin (CPT), which both generate 3'-end blocking DNA lesions that are also repaired by TDP1, we found that inactivation of BRCA2 renders cells hypersensitive to CNDAC and CPT but not to AraC. By contrast, cells lacking PARP1 were only hypersensitive to CPT but not to CNDAC or AraC. Examination of expression in the cancer cell line databases (CCLE, GDSC, NCI-60) and human cancers (TCGA) revealed a broad range of expression of , which was correlated with PARP1 expression, gene copy number and promoter methylation. Thus, this study identifies the importance of TDP1 as a novel determinant of response to CNDAC across various cancer types (especially non-small cell lung cancers), and demonstrates the differential involvement of BRCA2, PARP1, and TDP1 in the cellular responses to CNDAC, AraC, and CPT. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971110PMC
November 2017

The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C).

Oncotarget 2017 May;8(20):33457-33474

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-Ku, Kyoto 606-8501, Japan.

Chemotherapeutic nucleoside analogs, such as Ara-C, 5-Fluorouracil (5-FU) and Trifluridine (FTD), are frequently incorporated into DNA by the replicative DNA polymerases. However, it remains unclear how this incorporation kills cycling cells. There are two possibilities: Nucleoside analog triphosphates inhibit the replicative DNA polymerases, and/or nucleotide analogs mis-incorporated into genomic DNA interfere with the next round of DNA synthesis as replicative DNA polymerases recognize them as template DNA lesions, arresting synthesis. To address the first possibility, we selectively disrupted the proofreading exonuclease activity of DNA polymerase ε (Polε), the leading-strand replicative polymerase in avian DT40 and human TK6 cell lines. To address the second, we disrupted RAD18, a gene involved in translesion DNA synthesis, a mechanism that relieves stalled replication. Strikingly, POLE1exo-/- cells, but not RAD18-/- cells, were hypersensitive to Ara-C, while RAD18-/- cells were hypersensitive to FTD. gH2AX focus formation following a pulse of Ara-C was immediate and did not progress into the next round of replication, while gH2AX focus formation following a pulse of 5-FU and FTD was delayed to the next round of replication. Biochemical studies indicate that human proofreading-deficient Polε-exo- holoenzyme incorporates Ara-CTP, but subsequently extend from this base several times less efficiently than from intact nucleotides. Together our results suggest that Ara-C acts by blocking extension of the nascent DNA strand and is counteracted by the proofreading activity of Polε, while 5-FU and FTD are efficiently incorporated but act as replication fork blocks in the subsequent S phase, which is counteracted by translesion synthesis.
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http://dx.doi.org/10.18632/oncotarget.16508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464882PMC
May 2017

Cytotoxicity of Tirapazamine (3-Amino-1,2,4-benzotriazine-1,4-dioxide)-Induced DNA Damage in Chicken DT40 Cells.

Chem Res Toxicol 2017 02 27;30(2):699-704. Epub 2016 Dec 27.

Division of Radiation Life Science, Research Reactor Institute, Kyoto University , 2 Asashiro-Nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan.

Tirapazamine (TPZ) is an anticancer drug with highly selective cytotoxicity toward hypoxic cells. TPZ is converted to a radical intermediate under hypoxic conditions, and this intermediate interacts with intracellular macromolecules, including DNA. TPZ has been reported to indirectly induce DNA double-strand breaks (DSBs) through the formation of various intermediate DNA lesions under hypoxic conditions. Although the topoisomerase II-DNA complex has been identified as one of these intermediates, other lesions have not yet been defined. In order to obtain a deeper understanding of the mechanisms responsible for the selective cytotoxicity of TPZ toward hypoxic cells, its cellular sensitivity was systematically examined with genetically isogenic DNA-repair-deficient mutant DT40 cell lines. Our results showed that tdp1, tdp2, parp1, and aptx1 cells displayed hypersensitivity to TPZ only under hypoxic conditions. These results strongly suggest that the accumulation of the topoisomerase I-trapped DNA complex, topoisomerase II-trapped DNA complex, and abortive ligation products with 5'-AMP are the potential causes of TPZ-induced hypoxic cell death. Furthermore, our genetic analysis revealed that under normoxic conditions (as well as hypoxic conditions), TPZ exhibited significant cytotoxicity toward cell lines deficient in homologous recombination, nonhomologous end joining, base excision repair, and translesion synthesis. Ascorbic acid, a radical scavenger, suppressed TPZ-induced cytotoxicity toward normoxic cells. These results suggest the involvement of oxidative DNA damage and DSBs produced by reactive oxygen species generated from superoxide, a byproduct of the oxidation of TPZ radical intermediates in normoxic cells. Collectively, our results demonstrate that TPZ induces oxidative DNA damage under normoxic and hypoxic conditions and selectively introduces abortive topoisomerase-DNA complexes and unligatable DNA ends under hypoxic conditions.
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http://dx.doi.org/10.1021/acs.chemrestox.6b00417DOI Listing
February 2017
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