Publications by authors named "Shunichi Takeda"

224 Publications

PRDX1 is essential for the viability and maintenance of reactive oxygen species in chicken DT40.

Genes Environ 2021 Aug 5;43(1):35. Epub 2021 Aug 5.

Department of Biological Sciences, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599-8531, Japan.

Background: Peroxiredoxin 1 (PRDX1) is a member of a ubiquitous family of thiol peroxidases that catalyze the reduction of peroxides, including hydrogen peroxide. It functions as an antioxidant enzyme, similar to catalase and glutathione peroxidase. PRDX1 was recently shown act as a sensor of reactive oxygen species (ROS) and play a role in ROS-dependent intracellular signaling pathways. To investigate its physiological functions, PRDX1 was conditionally disrupted in chicken DT40 cells in the present study.

Results: The depletion of PRDX1 resulted in cell death with increased levels of intracellular ROS. PRDX1-depleted cells did not show the accumulation of chromosomal breaks or sister chromatid exchange (SCE). These results suggest that cell death in PRDX1-depleted cells was not due to DNA damage. 2-Mercaptoethanol protected against cell death in PRDX1-depleted cells and also suppressed elevations in ROS.

Conclusions: PRDX1 is essential in chicken DT40 cells and plays an important role in maintaining intracellular ROS homeostasis (or in the fine-tuning of cellular ROS levels). Cells deficient in PRDX1 may be used as an endogenously deregulated ROS model to elucidate the physiological roles of ROS in maintaining proper cell growth.
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http://dx.doi.org/10.1186/s41021-021-00211-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8340460PMC
August 2021

FANCD2-Associated Nuclease 1 Partially Compensates for the Lack of Exonuclease 1 in Mismatch Repair.

Mol Cell Biol 2021 08 24;41(9):e0030321. Epub 2021 Aug 24.

Institute of Molecular Cancer Research, University of Zurichgrid.7400.3, Zurich, Switzerland.

Germline mutations in the mismatch repair (MMR) genes , , , and are linked to cancer of the colon and other organs, characterized 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 to MMR ; cells lacking both EXO1 and FAN1 have an MMR defect and display resistance to -methyl--nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, suggesting 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8384067PMC
August 2021

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

Mutagenesis 2021 Jul 3. Epub 2021 Jul 3.

Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tonomachi, Kawasaki-ku, Kawasaki Japan.

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) that is deficient in DNA excision repair system using human lymphoblastoid TK6 cells lacking XRCC1 and XPA (XRCC1 -/-/XPA -/-), the core factors of base excision repair and nucleotide excision repair, 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 focused 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.
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http://dx.doi.org/10.1093/mutage/geab025DOI Listing
July 2021

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

Nat Struct Mol Biol 2021 06 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

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

PLoS One 2021 1;16(6):e0252587. Epub 2021 Jun 1.

Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Hachioji-shi, Tokyo, Japan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0252587PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168857PMC
June 2021

Impact of Gba2 on neuronopathic Gaucher's disease and α-synuclein accumulation in medaka (Oryzias latipes).

Mol Brain 2021 05 10;14(1):80. Epub 2021 May 10.

Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, 606-8507, Japan.

Homozygous mutations in the lysosomal glucocerebrosidase gene, GBA1, cause Gaucher's disease (GD), while heterozygous mutations in GBA1 are a strong risk factor for Parkinson's disease (PD), whose pathological hallmark is intraneuronal α-synuclein (asyn) aggregates. We previously reported that gba1 knockout (KO) medaka exhibited glucosylceramide accumulation and neuronopathic GD phenotypes, including short lifespan, the dopaminergic and noradrenergic neuronal cell loss, microglial activation, and swimming abnormality, with asyn accumulation in the brains. A recent study reported that deletion of GBA2, non-lysosomal glucocerebrosidase, in a non-neuronopathic GD mouse model rescued its phenotypes. In the present study, we generated gba2 KO medaka and examined the effect of Gba2 deletion on the phenotypes of gba1 KO medaka. The Gba2 deletion in gba1 KO medaka resulted in the exacerbation of glucosylceramide accumulation and no improvement in neuronopathic GD pathological changes, asyn accumulation, or swimming abnormalities. Meanwhile, though gba2 KO medaka did not show any apparent phenotypes, biochemical analysis revealed asyn accumulation in the brains. gba2 KO medaka showed a trend towards an increase in sphingolipids in the brains, which is one of the possible causes of asyn accumulation. In conclusion, this study demonstrated that the deletion of Gba2 does not rescue the pathological changes or behavioral abnormalities of gba1 KO medaka, and GBA2 represents a novel factor affecting asyn accumulation in the brains.
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http://dx.doi.org/10.1186/s13041-021-00790-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8111776PMC
May 2021

Critical roles of Rad54 in tolerance to apigenin-induced Top1-mediated DNA damage.

Exp Ther Med 2021 May 18;21(5):505. Epub 2021 Mar 18.

Department of Pharmacology, Key Laboratory of Drug-Targeting and Drug Delivery Systems of the Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, P.R. China.

Apigenin (APG), a flavone sub-class of flavonoids, possesses a diverse range of biological activities, including anti-cancer and anti-inflammatory effects. Previous studies identified the genotoxicity of APG in certain cancer cells, which may be associated with its anticancer effect. However, the DNA damage repair mechanism induced by APG has remained elusive. In order to clarify the molecular mechanisms, the present study determined the toxicity of APG to the wild-type () DT40 chicken B-lymphocyte cell line, as well as to DT40 cells with deletions in various DNA repair genes, and their sensitivities were compared. It was demonstrated that cells deficient of Rad54, a critical homologous recombination gene, were particularly sensitive to APG. Cell-cycle analysis demonstrated that APG caused an increase in the G/M-phase population of cells that was greater than that in cells. Furthermore, it was demonstrated by immunofluorescence assay that cells exhibited significantly increased numbers of γ-phosphorylated H2AX variant histone foci and chromosomal aberrations compared to the cells in response to APG. Of note, the complex of enzyme assay indicated that APG induced increased topoisomerase I (Top1) covalent protein DNA complex in cells compared to cells. Finally, these results were verified using the TK6 human lymphoblastoid cell line and it was demonstrated that, as for DT40 cells, Rad54 deficiency sensitized TK6 cells to APG. The present study demonstrated that Rad54 was involved in the repair of APG-induced DNA damage, which was associated with Top1 inhibition.
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http://dx.doi.org/10.3892/etm.2021.9936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005727PMC
May 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

Active learning effectively identifies a minimal set of maximally informative and asymptotically performant cytotoxic structure-activity patterns in NCI-60 cell lines.

RSC Med Chem 2020 Sep 20;11(9):1075-1087. Epub 2020 Jul 20.

Kyoto University Graduate School of Medicine , Department of Molecular Biosciences , Life Science Informatics Research Unit , Konoemachi Yoshida Sakyo , Kyoto 606-8501 , Japan . Email:

The NCI-60 cancer cell line screening panel has provided insights for development of subtype-specific chemical therapies and repurposing. By extracting chemical structure and cytotoxicity patterns, virtual screening potentially complements the availability of high-throughput assay platforms and improves bioactive compound discovery rates by computational prefiltering of candidate compound libraries. Many groups report high prediction performances in computational models of NCI-60 data when using cross-validation or similar techniques, yet prospective therapy development in novel cancers may have little to no such data and further may not have the resources to perform hit identification using large compound libraries. In contrast to bulk screening and analysis, the active learning methodology has demonstrated how to identify compounds for screening in small batches and update computational models iteratively, leading to predictive models with a minimum number of compounds, and importantly clarifying data volumes at which limits in predictive ability are achieved. Here, in replicate per-cell line experiments using 50% of data (∼20 000 compounds) as the external prediction target, predictive limits are reproducibly demonstrated at the stage of systematic selection of 10-30% of the incorporable half. The pattern was consistent across all 60 cell lines. Limits of predictability are found to be correlated to the doubling times of cell lines and the number of cellular response discontinuities (activity cliffs) present per cell line. Organization into chemical scaffolds delineated degrees of predictive challenge. These results provide key insights for strategies in developing new inhibitors in existing cell lines or for future automated therapy selection in personalized oncotherapy.
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http://dx.doi.org/10.1039/d0md00110dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513593PMC
September 2020

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

RAD52 Adjusts Repair of Single-Strand Breaks via Reducing DNA-Damage-Promoted XRCC1/LIG3α Co-localization.

Cell Rep 2021 01;34(2):108625

Department of Radiation Oncology, Emory University School of Medicine and the Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA. Electronic address:

Radiation sensitive 52 (RAD52) is an important factor for double-strand break repair (DSBR). However, deficiency in vertebrate/mammalian Rad52 has no apparent phenotype. The underlying mechanism remains elusive. Here, we report that RAD52 deficiency increased cell survival after camptothecin (CPT) treatment. CPT generates single-strand breaks (SSBs) that further convert to double-strand breaks (DSBs) if they are not repaired. RAD52 inhibits SSB repair (SSBR) through strong single-strand DNA (ssDNA) and/or poly(ADP-ribose) (PAR) binding affinity to reduce DNA-damage-promoted X-Ray Repair Cross Complementing 1 (XRCC1)/ligase IIIα (LIG3α) co-localization. The inhibitory effects of RAD52 on SSBR neutralize the role of RAD52 in DSBR, suggesting that RAD52 may maintain a balance between cell survival and genomic integrity. Furthermore, we demonstrate that blocking RAD52 oligomerization that disrupts RAD52's DSBR, while retaining its ssDNA binding capacity that is required for RAD52's inhibitory effects on SSBR, sensitizes cells to different DNA-damaging agents. This discovery provides guidance for developing efficient RAD52 inhibitors in cancer therapy.
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http://dx.doi.org/10.1016/j.celrep.2020.108625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872142PMC
January 2021

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

A Surge of DNA Damage Links Transcriptional Reprogramming and Hematopoietic Deficit in Fanconi Anemia.

Mol Cell 2020 12;80(6):1013-1024.e6

Department of Experimental Radiation Oncology, the University of Texas, MD Anderson Cancer, Houston, TX 77030, USA; Life Sciences Institute, Zhejiang University, Hangzhou 310058, China. Electronic address:

Impaired DNA crosslink repair leads to Fanconi anemia (FA), characterized by a unique manifestation of bone marrow failure and pancytopenia among diseases caused by DNA damage response defects. As a germline disorder, why the hematopoietic hierarchy is specifically affected is not fully understood. We find that reprogramming transcription during hematopoietic differentiation results in an overload of genotoxic stress, which causes aborted differentiation and depletion of FA mutant progenitor cells. DNA damage onset most likely arises from formaldehyde, an obligate by-product of oxidative protein demethylation during transcription regulation. Our results demonstrate that rapid and extensive transcription reprogramming associated with hematopoietic differentiation poses a major threat to genome stability and cell viability in the absence of the FA pathway. The connection between differentiation and DNA damage accumulation reveals a novel mechanism of genome scarring and is critical to exploring therapies to counteract the aplastic anemia for the treatment of FA patients.
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http://dx.doi.org/10.1016/j.molcel.2020.11.040DOI Listing
December 2020

The fragility of a structurally diverse duplication block triggers recurrent genomic amplification.

Nucleic Acids Res 2021 01;49(1):244-256

Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.

The human genome contains hundreds of large, structurally diverse blocks that are insufficiently represented in the reference genome and are thus not amenable to genomic analyses. Structural diversity in the human population suggests that these blocks are unstable in the germline; however, whether or not these blocks are also unstable in the cancer genome remains elusive. Here we report that the 500 kb block called KRTAP_region_1 (KRTAP-1) on 17q12-21 recurrently demarcates the amplicon of the ERBB2 (HER2) oncogene in breast tumors. KRTAP-1 carries numerous tandemly-duplicated segments that exhibit diversity within the human population. We evaluated the fragility of the block by cytogenetically measuring the distances between the flanking regions and found that spontaneous distance outliers (i.e DNA breaks) appear more frequently at KRTAP-1 than at the representative common fragile site (CFS) FRA16D. Unlike CFSs, KRTAP-1 is not sensitive to aphidicolin. The exonuclease activity of DNA repair protein Mre11 protects KRTAP-1 from breaks, whereas CtIP does not. Breaks at KRTAP-1 lead to the palindromic duplication of the ERBB2 locus and trigger Breakage-Fusion-Bridge cycles. Our results indicate that an insufficiently investigated area of the human genome is fragile and could play a crucial role in cancer genome evolution.
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http://dx.doi.org/10.1093/nar/gkaa1136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797068PMC
January 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

Critical roles of tyrosyl-DNA phosphodiesterases in cell tolerance to carnosol-induced DNA damage.

Cell Biol Int 2020 Aug 28;44(8):1640-1650. Epub 2020 Apr 28.

Department of Pharmacology, Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, China.

Carnosol is a natural compound with pharmacological action due to its anti-cancer properties. However, the precise mechanism for its anti-carcinogenic effect remains elusive. In this study, we used lymphoblastoid TK6 cell lines to identify the DNA damage and repair mechanisms of carnosol. Our results showed that carnosol induced DNA double-strand breaks (DSBs). We also found that cells lacking tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme related to topoisomerase 1 (TOP1), and tyrosyl-DNA phosphodiesterase 2 (TDP2), an enzyme related to topoisomerase 2 (TOP2), were supersensitive to carnosol. Carnosol was found to induce the formation of the TOP1-DNA cleavage complex (TOP1cc) and TOP2-DNA cleavage complex (TOP2cc). When comparing the accumulation of γ-H2AX foci and the number of chromosomal aberrations (CAs) with wild-type (WT) cells, the susceptivity of the TDP1 and TDP2 cells were associated with an increased DNA damage. Our results provided evidence of carnosol inducing DNA lesions in TK6 cells and demonstrated that the damage induced by carnosol was associated with abnormal topoisomerase activity. We conclude that TDP1 and TDP2 play important roles in the anti-cancer effect of carnosol.
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http://dx.doi.org/10.1002/cbin.11357DOI Listing
August 2020

ATAD5 deficiency alters DNA damage metabolism and sensitizes cells to PARP inhibition.

Nucleic Acids Res 2020 05;48(9):4928-4939

Institute of Molecular Life Sciences of the University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

Replication factor C (RFC), a heteropentamer of RFC1-5, loads PCNA onto DNA during replication and repair. Once DNA synthesis has ceased, PCNA must be unloaded. Recent findings assign the uloader role primarily to an RFC-like (RLC) complex, in which the largest RFC subunit, RFC1, has been replaced with ATAD5 (ELG1 in Saccharomyces cerevisiae). ATAD5-RLC appears to be indispensable, given that Atad5 knock-out leads to embryonic lethality. In order to learn how the retention of PCNA on DNA might interfere with normal DNA metabolism, we studied the response of ATAD5-depleted cells to several genotoxic agents. We show that ATAD5 deficiency leads to hypersensitivity to methyl methanesulphonate (MMS), camptothecin (CPT) and mitomycin C (MMC), agents that hinder the progression of replication forks. We further show that ATAD5-depleted cells are sensitive to poly(ADP)ribose polymerase (PARP) inhibitors and that the processing of spontaneous oxidative DNA damage contributes towards this sensitivity. We posit that PCNA molecules trapped on DNA interfere with the correct metabolism of arrested replication forks, phenotype reminiscent of defective homologous recombination (HR). As Atad5 heterozygous mice are cancer-prone and as ATAD5 mutations have been identified in breast and endometrial cancers, our finding may open a path towards the therapy of these tumours.
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http://dx.doi.org/10.1093/nar/gkaa255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229844PMC
May 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

Nonhomologous end joining and homologous recombination involved in luteolin-induced DNA damage in DT40 cells.

Toxicol In Vitro 2020 Jun 10;65:104825. Epub 2020 Mar 10.

Department of Pharmacology, Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, China. Electronic address:

Luteolin (3',4',5,7-tetrahydroxyflavone), a naturally occurring flavonoid, has been shown to have anticancer activity in many types of cancer cell lines. The anticancer capacity of luteolin may be related to its ability to induce DNA double-strand breaks (DSBs). Here, we used DT40 cells to determine whether nonhomologous end joining (NHEJ) and homologous recombination (HR) are involved in the repair mechanism of luteolin-induced DNA damage. Cells defective in Ku70 (an enzyme associated with NHEJ) or Rad54 (an enzyme essential for HR) were hypersensitive and presented more apoptosis in response to luteolin. Moreover, the sensitivity and apoptosis of Ku70 and Rad54 cells were associated with increased DNA damage when the numbers of γ-H2AX foci and chromosomal aberrations (CAs) were compared with those from WT cells. Additionally, after treatment with luteolin, Ku70 cells presented more Top2 covalent cleavage complexes (Top2cc). These results indicated that luteolin induced DSBs in DT40 cells and demonstrated that both NHEJ and HR participated in the repair of luteolin-induced DSBs, which might be related to the inhibition of topoisomerases. These results imply that simultaneous inhibition of NHEJ and HR with luteolin treatment would provide a powerful protocol in cancer chemotherapy.
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http://dx.doi.org/10.1016/j.tiv.2020.104825DOI Listing
June 2020

The ARK Assay Is a Sensitive and Versatile Method for the Global Detection of DNA-Protein Crosslinks.

Cell Rep 2020 01;30(4):1235-1245.e4

Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA. Electronic address:

DNA-protein crosslinks (DPCs) are a frequent form of DNA lesion and are strongly inhibitive in diverse DNA transactions. Despite recent developments, the biochemical detection of DPCs remains a limiting factor for the in-depth mechanistic understanding of DPC repair. Here, we develop a sensitive and versatile assay, designated ARK, for the quantitative analysis of DPCs in cells. ARK uses sequential chaotropic and detergent-based isolation of DPCs and substantially enhances sample purity, resulting in a 5-fold increase in detection sensitivity and a 10-fold reduction in background reading. We validate the ARK assay with genetic mutants with established deficiencies in DPC repair and demonstrate its robustness by using common DPC-inducing reagents, including formaldehyde, camptothecin, and etoposide. In addition, we show that the Fanconi anemia pathway contributes to the repair of DPCs. Thus, ARK is expected to facilitate various studies aimed at understanding both fundamental biology and translational applications of DNA-protein crosslink repair.
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http://dx.doi.org/10.1016/j.celrep.2019.12.067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069250PMC
January 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

Correlation of homologous recombination deficiency induced mutational signatures with sensitivity to PARP inhibitors and cytotoxic agents.

Genome Biol 2019 11 14;20(1):240. Epub 2019 Nov 14.

Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Magyar tudosok krt 2, Budapest, H-1117, Hungary.

Background: Homologous recombination (HR) repair deficiency arising from defects in BRCA1 or BRCA2 is associated with characteristic patterns of somatic mutations. In this genetic study, we ask whether inactivating mutations in further genes of the HR pathway or the DNA damage checkpoint also give rise to somatic mutation patterns that can be used for treatment prediction.

Results: Using whole genome sequencing of an isogenic knockout cell line panel, we find a universal HR deficiency-specific base substitution signature that is similar to COSMIC signature 3. In contrast, we detect different deletion phenotypes corresponding to specific HR mutants. The inactivation of BRCA2 or PALB2 leads to larger deletions, typically with microhomology, when compared to the disruption of BRCA1, RAD51 paralogs, or RAD54. Comparison with the deletion spectrum of Cas9 cut sites suggests that most spontaneously arising genomic deletions are not the consequence of double-strand breaks. Surprisingly, the inactivation of checkpoint kinases ATM and CHK2 has no mutagenic consequences. Analysis of tumor exomes with biallelic inactivating mutations in the investigated genes confirms the validity of the cell line models. We present a comprehensive analysis of sensitivity of the investigated mutants to 13 therapeutic agents for the purpose of correlating genomic mutagenic phenotypes with drug sensitivity.

Conclusion: Our results suggest that no single genomic mutational class shows perfect correlation with sensitivity to common treatments, but the contribution of COSMIC signature 3 to base substitutions, or a combined measure of different features, may be reasonably good at predicting platinum and PARP inhibitor sensitivity.
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http://dx.doi.org/10.1186/s13059-019-1867-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857305PMC
November 2019

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

Applicability Domain of Active Learning in Chemical Probe Identification: Convergence in Learning from Non-Specific Compounds and Decision Rule Clarification.

Molecules 2019 Jul 26;24(15). Epub 2019 Jul 26.

Kyoto University Graduate School of Medicine, Department of Molecular Biosciences, Life Science Informatics Research Unit, Kyoto, Sakyo, Yoshida, Konoemachi, Kyoto 606-8501, Japan.

Efficient identification of chemical probes for the manipulation and understanding of biological systems demands specificity for target proteins. Computational means to optimize candidate compound selection for experimental selectivity evaluation are being sought. The active learning virtual screening method has demonstrated the ability to efficiently converge on predictive models with reduced datasets, though its applicability domain to probe identification has yet to be determined. In this article, we challenge active learning's ability to predict inhibitory bioactivity profiles of selective compounds when learning from chemogenomic features found in non-selective ligand-target pairs. Comparison of controls versus multiple molecule representations de-convolutes factors contributing to predictive capability. Experiments using the matrix metalloproteinase family demonstrate maximum probe bioactivity prediction achieved from only approximately 20% of non-probe bioactivity; this data volume is consistent with prior chemogenomic active learning studies despite the increased difficulty from chemical biology experimental settings used here. Feature weight analyses are combined with a custom visualization to unambiguously detail how active learning arrives at classification decisions, yielding clarified expectations for chemogenomic modeling. The results influence tactical decisions for computational probe design and discovery.
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http://dx.doi.org/10.3390/molecules24152716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6696588PMC
July 2019
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