title: BRCA1 and CtIP Are Both Required to Recruit Dna2 at Double-Strand Breaks in Homologous Recombination.
abstract: Homologous recombination plays a key role in the repair of double-strand breaks (DSBs), and thereby significantly contributes to cellular tolerance to radiotherapy and some chemotherapy. DSB repair by homologous recombination is initiated by 5' to 3' strand resection (DSB resection), with nucleases generating the 3' single-strand DNA (3'ssDNA) at DSB sites. Genetic studies of Saccharomyces cerevisiae demonstrate a two-step DSB resection, wherein CtIP and Mre11 nucleases carry out short-range DSB resection followed by long-range DSB resection done by Dna2 and Exo1 nucleases. Recent studies indicate that CtIP contributes to DSB resection through its non-catalytic role but not as a nuclease. However, it remains elusive how CtIP contributes to DSB resection. To explore the non-catalytic role, we examined the dynamics of Dna2 by developing an immuno-cytochemical method to detect ionizing-radiation (IR)-induced Dna2-subnuclear-focus formation at DSB sites in chicken DT40 and human cell lines. Ionizing-radiation induced Dna2 foci only in wild-type cells, but not in Dna2 depleted cells, with the number of foci reaching its maximum at 30 minutes and being hardly detectable at 120 minutes after IR. Induced foci were detectable in cells in the G2 phase but not in the G1 phase. These observations suggest that Dna2 foci represent the recruitment of Dna2 to DSB sites for DSB resection. Importantly, the depletion of CtIP inhibited the recruitment of Dna2 to DSB sites in both human cells and chicken DT40 cells. Likewise, a defect in breast cancer 1 (BRCA1), which physically interacts with CtIP and contributes to DSB resection, also inhibited the recruitment of Dna2. Moreover, CtIP physically associates with Dna2, and the association is enhanced by IR. We conclude that BRCA1 and CtIP contribute to DSB resection by recruiting Dna2 to damage sites, thus ensuring the robust DSB resection necessary for efficient homologous recombination.
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto, Japan.
authors.forename: Nguyen Ngoc
authors.initials: NN
authors.lastname: Hoa
authors.affiliation: Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Kobayashi
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Omura
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Hirakawa
authors.affiliation: The Howard Hughes Medical Institute, The Department of Molecular Biosciences, and The Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, United States of America.
authors.forename: Soo-Hyun
authors.initials: SH
authors.lastname: Yang
authors.affiliation: Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto, Japan.
authors.forename: Kenshi
authors.initials: K
authors.lastname: Komatsu
authors.affiliation: The Howard Hughes Medical Institute, The Department of Molecular Biosciences, and The Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, United States of America.
authors.forename: Tanya T
authors.initials: TT
authors.lastname: Paull
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto, Japan.
authors.forename: Shunichi
authors.initials: S
authors.lastname: Takeda
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-ku, Kyoto, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sasanuma
mesh_terms: D000818:Animals; D019313:BRCA1 Protein / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D059447:Cell Cycle Checkpoints / Q000235:genetics; D002460:Cell Line; D002645:Chickens; D002869:Chromosome Aberrations; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases; D004789:Enzyme Activation; D004843:Epistasis, Genetic; D055879:Gene Knock-In Techniques; D059765:Homologous Recombination; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D051135:Rad51 Recombinase / Q000378:metabolism
chemical_list: D019313:BRCA1 Protein; D002352:Carrier Proteins; D009687:Nuclear Proteins; D051135:Rad51 Recombinase; D004706:Endodeoxyribonucleases; C410741:RBBP8 protein, human; D004265:DNA Helicases; C509908:DNA2 protein, human