UV-induced ataxia-telangiectasia-mutated and Rad3-related (ATR) activation requires replication stress.
Ataxia-telangiectasia-mutated and Rad3-related (ATR) plays an essential role in the maintenance of genome integrity and cell viability. The kinase is activated in response to DNA damage and initiates a checkpoint signaling cascade by phosphorylating a number of downstream substrates including Chk1. Unlike ataxia-telangiectasia-mutated (ATM), which appears to be mainly activated by DNA double-strand breaks, ATR can be activated by a variety of DNA damaging agents. However, it is still unclear what triggers ATR activation in response to such diverse DNA lesions. One model proposes that ATR can directly recognize DNA lesions, while other recent data suggest that ATR is activated by a common single-stranded DNA (ssDNA) intermediate generated during DNA repair. In this study, we show that UV lesions do not directly activate ATR in vivo. In addition, ssDNA lesions created during the repair of UV damage are also not sufficient to activate the ATR-dependent pathway. ATR activation is only observed in replicating cells indicating that replication stress is required to trigger the ATR-mediated checkpoint cascade in response to UV irradiation. Interestingly, H2AX appears to be required for the accumulation of ATR at stalled replication forks. Together our data suggest that ssDNA at arrested replication forks recruits ATR and initiates ATR-mediated phosphorylation of H2AX and Chk1. Phosphorylated H2AX might further facilitate ATR activation by stabilizing ATR at the sites of arrested replication forks.
Ward
Irene M
IM
Guggenheim 1306, Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905, USA.
Minn
Kay
K
Chen
Junjie
J
D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002469:Cell Separation; D002470:Cell Survival; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004277:DNA, Single-Stranded; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D015398:Signal Transduction; D013312:Stress, Physiological; D013997:Time Factors; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1