DNA-PKcs and ATM co-regulate DNA double-strand break repair.
DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and approximately 4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR.
Shrivastav
Meena
M
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA.
Miller
Cheryl A
CA
De Haro
Leyma P
LP
Durant
Stephen T
ST
Chen
Benjamin P C
BP
Chen
David J
DJ
Nickoloff
Jac A
JA
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000528:radiation effects; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000528:radiation effects; D010769:Phosphothreonine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000378:metabolism; D011839:Radiation, Ionizing; D011995:Recombination, Genetic / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D010769:Phosphothreonine; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D051135:Rad51 Recombinase