title: Perturbed gap-filling synthesis in nucleotide excision repair causes histone H2AX phosphorylation in human quiescent cells.
abstract: Human histone H2AX is rapidly phosphorylated on serine 139 in response to DNA double-strand breaks and plays a crucial role in tethering the factors involved in DNA repair and damage signaling. Replication stress caused by hydroxyurea or UV also initiates H2AX phosphorylation in S-phase cells, although UV-induced H2AX phosphorylation in non-cycling cells has recently been observed. Here we study the UV-induced H2AX phosphorylation in human primary fibroblasts under growth-arrested conditions. This reaction absolutely depends on nucleotide excision repair (NER) and is mechanistically distinct from the replication stress-induced phosphorylation. The treatment of cytosine-beta-D-arabinofuranoside strikingly enhances the NER-dependent H2AX phosphorylation and induces the accumulation of replication protein A (RPA) and ATR-interacting protein (ATRIP) at locally UV-damaged subnuclear regions. Consistently, the phosphorylation appears to be mainly mediated by ataxia-telangiectasia mutated and Rad3-related (ATR), although Chk1 (Ser345) is not phosphorylated by the activated ATR. The cellular levels of DNA polymerases delta and epsilon and proliferating cell nuclear antigen are markedly reduced in quiescent cells. We propose a model that perturbed gap-filling synthesis following dual incision in NER generates single-strand DNA gaps and hence initiates H2AX phosphorylation by ATR with the aid of RPA and ATRIP.
authors.affiliation: Laboratory of Human Molecular Genetics, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Matsumoto
authors.forename: Kie
authors.initials: K
authors.lastname: Yaginuma
authors.forename: Ai
authors.initials: A
authors.lastname: Igarashi
authors.forename: Mayumi
authors.initials: M
authors.lastname: Imura
authors.forename: Mizuho
authors.initials: M
authors.lastname: Hasegawa
authors.forename: Kuniyoshi
authors.initials: K
authors.lastname: Iwabuchi
authors.forename: Takayasu
authors.initials: T
authors.lastname: Date
authors.forename: Toshio
authors.initials: T
authors.lastname: Mori
authors.forename: Kanji
authors.initials: K
authors.lastname: Ishizaki
authors.forename: Katsumi
authors.initials: K
authors.lastname: Yamashita
authors.forename: Manabu
authors.initials: M
authors.lastname: Inobe
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Matsunaga
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D003561:Cytarabine / Q000494:pharmacology; D004249:DNA Damage / Q000502:physiology / Q000528:radiation effects; D004257:DNA Polymerase II / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000502:physiology*; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D006657:Histones / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014466:Ultraviolet Rays / Q000009:adverse effects
chemical_list: C521194:ATRIP protein, human; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D003561:Cytarabine; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D004257:DNA Polymerase II; D004258:DNA Polymerase III