title: Collaborative roles of gammaH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair.
abstract: One of the earliest events in the signal transduction cascade that initiates a DNA damage checkpoint is the phosphorylation on serine 139 of histone H2AX (gammaH2AX) at DNA double-strand breaks (DSBs). However, the role of gammaH2AX in DNA repair is poorly understood. To address this question, we generated chicken DT40 cells carrying a serine to alanine mutation at position 139 of H2AX (H2AX(-/S139A)) and examined their DNA repair capacity. H2AX(-/S139A) cells exhibited defective homologous recombinational repair (HR) as manifested by delayed Rad51 focus formation following ionizing radiation (IR) and hypersensitivity to the topoisomerase I inhibitor, camptothecin (CPT), which causes DSBs at replication blockage. Deletion of the Rad51 paralog gene, XRCC3, also delays Rad51 focus formation. To test the interaction of Xrcc3 and gammaH2AX, we disrupted XRCC3 in H2AX(-/S139A) cells. XRCC3(-/-)/H2AX(-/S139A) mutants were not viable, although this synthetic lethality was reversed by inserting a transgene that conditionally expresses wild-type H2AX. Upon repression of the wild-type H2AX transgene, XRCC3(-/-)/H2AX(-/S139A) cells failed to form Rad51 foci and exhibited markedly increased levels of chromosomal aberrations after CPT treatment. These results indicate that H2AX and XRCC3 act in separate arms of a branched pathway to facilitate Rad51 assembly.
authors.affiliation: CREST Laboratory, Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan.
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Sonoda
authors.forename: Guang Yu
authors.initials: GY
authors.lastname: Zhao
authors.forename: Masaoki
authors.initials: M
authors.lastname: Kohzaki
authors.forename: Pawan Kumar
authors.initials: PK
authors.lastname: Dhar
authors.forename: Koji
authors.initials: K
authors.lastname: Kikuchi
authors.forename: Christophe
authors.initials: C
authors.lastname: Redon
authors.forename: Duane R
authors.initials: DR
authors.lastname: Pilch
authors.forename: William M
authors.initials: WM
authors.lastname: Bonner
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nakano
authors.forename: Masami
authors.initials: M
authors.lastname: Watanabe
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Nakayama
authors.forename: Shunichi
authors.initials: S
authors.lastname: Takeda
authors.forename: Yasunari
authors.initials: Y
authors.lastname: Takami
mesh_terms: D000818:Animals; D030161:Avian Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002166:Camptothecin / Q000494:pharmacology; D002478:Cells, Cultured; D002645:Chickens; D004249:DNA Damage / Q000502:physiology; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005720:Gamma Rays; D042822:Genomic Instability; D006657:Histones / Q000235:genetics / Q000502:physiology*; D008957:Models, Genetic; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014162:Transfection
chemical_list: D030161:Avian Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006657:Histones; C110901:X-ray repair cross complementing protein 3; D051135:Rad51 Recombinase; D002166:Camptothecin