Recruitment of DNA damage checkpoint proteins to damage in transcribed and nontranscribed sequences.
We developed a chromatin immunoprecipitation method for analyzing the binding of repair and checkpoint proteins to DNA base lesions in any region of the human genome. Using this method, we investigated the recruitment of DNA damage checkpoint proteins RPA, Rad9, and ATR to base damage induced by UV and acetoxyacetylaminofluorene in transcribed and nontranscribed regions in wild-type and excision repair-deficient human cells in G1 and S phases of the cell cycle. We find that all 3 damage sensors tested assemble at the site or in the vicinity of damage in the absence of DNA replication or repair and that transcription enhances recruitment of checkpoint proteins to the damage site. Furthermore, we find that UV irradiation of human cells defective in excision repair leads to phosphorylation of Chk1 kinase in both G1 and S phase of the cell cycle, suggesting that primary DNA lesions as well as stalled transcription complexes may act as signals to initiate the DNA damage checkpoint response.
Jiang
Guochun
G
Department of Biochemistry and Biophysics, Mary Ellen Jones Building CB 7260, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Sancar
Aziz
A
D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D047369:Chromatin Immunoprecipitation / Q000379:methods; D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics; D004307:Dose-Response Relationship, Radiation; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051716:Replication Protein A / Q000378:metabolism*; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D051716:Replication Protein A; C069930:rad9 protein; D004247:DNA; 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