title: ATR and H2AX cooperate in maintaining genome stability under replication stress.
abstract: Chromosomal abnormalities are frequently caused by problems encountered during DNA replication. Although the ATR-Chk1 pathway has previously been implicated in preventing the collapse of stalled replication forks into double-strand breaks (DSB), the importance of the response to fork collapse in ATR-deficient cells has not been well characterized. Herein, we demonstrate that, upon stalled replication, ATR deficiency leads to the phosphorylation of H2AX by ATM and DNA-PKcs and to the focal accumulation of Rad51, a marker of homologous recombination and fork restart. Because H2AX has been shown to play a facilitative role in homologous recombination, we hypothesized that H2AX participates in Rad51-mediated suppression of DSBs generated in the absence of ATR. Consistent with this model, increased Rad51 focal accumulation in ATR-deficient cells is largely dependent on H2AX, and dual deficiencies in ATR and H2AX lead to synergistic increases in chromatid breaks and translocations. Importantly, the ATM and DNA-PK phosphorylation site on H2AX (Ser(139)) is required for genome stabilization in the absence of ATR; therefore, phosphorylation of H2AX by ATM and DNA-PKcs plays a pivotal role in suppressing DSBs during DNA synthesis in instances of ATR pathway failure. These results imply that ATR-dependent fork stabilization and H2AX/ATM/DNA-PKcs-dependent restart pathways cooperatively suppress double-strand breaks as a layered response network when replication stalls.
authors.affiliation: Abramson Family Cancer Research Institute and Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Chanoux
authors.forename: Bu
authors.initials: B
authors.lastname: Yin
authors.forename: Karen A
authors.initials: KA
authors.lastname: Urtishak
authors.forename: Amma
authors.initials: A
authors.lastname: Asare
authors.forename: Craig H
authors.initials: CH
authors.lastname: Bassing
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Brown
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects; D042822:Genomic Instability; D006657:Histones / Q000502:physiology*; D008677:Metaphase; D051379:Mice; D018345:Mice, Knockout; D008938:Mitosis; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051135:Rad51 Recombinase / Q000378:metabolism; D011839:Radiation, Ionizing; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase / Q000502:physiology; D032681:Spectral Karyotyping; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051135:Rad51 Recombinase; D025521:Tumor Suppressor Proteins; C576897:Atr protein, mouse; C576325:Atm protein, mouse; C497264:Prkdc protein, mouse; C495154:Rad51 protein, mouse; C475961:gamma-H2AX protein, mouse