title: H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes.
abstract: DNA double-strand breaks (DSBs) are generated by the recombination activating gene (RAG) endonuclease in all developing lymphocytes as they assemble antigen receptor genes. DNA cleavage by RAG occurs only at the G1 phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening before their repair by classical non-homologous end-joining (NHEJ). Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently. Here, in vivo, we show that in murine cells the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1 (mediator of DNA damage checkpoint 1), which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ataxia telangiectasia mutated (ATM) kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle, in which it is essential for homology-mediated repair. In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and show significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes, thereby preventing these DNA ends from accessing repair pathways that promote genomic instability.
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri 63110, USA.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Helmink
authors.forename: Anthony T
authors.initials: AT
authors.lastname: Tubbs
authors.forename: Yair
authors.initials: Y
authors.lastname: Dorsett
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Bednarski
authors.forename: Laura M
authors.initials: LM
authors.lastname: Walker
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Feng
authors.forename: Girdhar G
authors.initials: GG
authors.lastname: Sharma
authors.forename: Peter J
authors.initials: PJ
authors.lastname: McKinnon
authors.forename: Junran
authors.initials: J
authors.lastname: Zhang
authors.forename: Craig H
authors.initials: CH
authors.lastname: Bassing
authors.forename: Barry P
authors.initials: BP
authors.lastname: Sleckman
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002461:Cell Line, Transformed; D002843:Chromatin / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004720:Endonucleases; D016193:G1 Phase; D015322:Gene Rearrangement, B-Lymphocyte / Q000235:genetics; D042822:Genomic Instability; D006657:Histones / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins; D054448:Precursor Cells, B-Lymphoid / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D013379:Substrate Specificity; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D004720:Endonucleases; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D025521:Tumor Suppressor Proteins; C501070:RBBP8 protein, mouse; C508937:MDC1 protein, mouse; C576325:Atm protein, mouse; C473337:Dclre1c protein, mouse; C475961:gamma-H2AX protein, mouse