ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses

Abstract

Genotoxic stress triggers the activation of checkpoints that delay cell-cycle progression to allow for DNA repair. Studies in fission yeast implicate members of the Rad family of checkpoint proteins, which includes Rad17, Rad1, Rad9 and Hus1, as key early-response elements during the activation of both the DNA damage and replication checkpoints. Here we demonstrate a direct regulatory linkage between the human Rad17 homologue (hRad17) and the checkpoint kinases, ATM and ATR. Treatment of human cells with genotoxic agents induced ATM/ATR-dependent phosphorylation of hRad17 at Ser 635 and Ser 645. Overexpression of a hRad17 mutant (hRad17AA) bearing Ala substitutions at both phosphorylation sites abrogated the DNA-damage-induced G2 checkpoint, and sensitized human fibroblasts to genotoxic stress. In contrast to wild-type hRad17, the hRad17AA mutant showed no ionizing-radiation-inducible association with hRad1, a component of the hRad1-hRad9-hHus1 checkpoint complex. These findings demonstrate that ATR/ATM-dependent phosphorylation of hRad17 is a critical early event during checkpoint signalling in DNA-damaged cells.

Regulation and mechanisms of mammalian double-strand break repair

“…It is generally believed that ATR is important for sensing UV and other types of bulky damage, whereas ATM is the damage sensor for IR-and radiomimetic drug-induced DNA damage. ATR, however, serves as a backup for ATM (Abraham, 2001). Furthermore, ATR appears to be the primary sensor of replicative blockade resulting from DNA lesions other than DSBs or by mechanisms other than direct DNA damage such as those occurring in response to DNA synthesis inhibitors.…”

“…In addition to these factors known to be directly involved in HRR, there are a number of molecules required for triggering DNA damage stress responses that act as 'sensors' for damage that are also important in cell cycle regulation and perhaps in the repair process itself. These sensors include ATM, ATR and DNA-PKcs (for recent reviews, see Abraham, 2001;Durocher and Jackson, 2001).…”

“…In addition, DSBs are potent triggers of cell cycle arrest and apoptosis under certain circumstances (Jackson, 2001;Norbury and Hickson, 2001). Specific cellular surveillance proteins appear to determine cell fate and the balance between cell cycle arrest, DNA repair and apoptosis (Dasika et al, 1999;Bernstein et al, 2002), such as the protein mutated in ataxia telangiectasia (ATM), ATM-and RAD3-related (ATR), and DNA-PK (Abraham, 2001;Durocher and Jackson, 2001). In addition, the tumor suppressors P53, BRCA1, and BRCA2 are also important for DSB repair (Khanna and Jackson, 2001), and all three have broad effects on cellular physiology and genomic stability.…”

hSMG-1 and ATM sequentially and independently regulate the G1 checkpoint during oxidative stress

“…Activated PIKKs phosphorylate various downstream targets involved in controlling cell cycle checkpoints, DNA repair and apoptotic pathways. These include p53, Chk1, Chk2, Brca-1, Rad9, Rad17, H2AX and others (Canman et al, 1998;Cortez et al, 1999;Bao et al, 2001;Chen et al, 2001;Bartek and Lukas, 2003;Stiff et al, 2004). Of particular importance to checkpoint activation is p53 phosphorylation on serine 15.…”

HSV-1 amplicon vector-mediated expression of ATM cDNA and correction of the ataxia–telangiectasia cellular phenotype

“…For example, phosphorylation of p53, Mdm2, and Chk2 participate in the G 1 checkpoint; [11][12][13] Nbs1, Brca1, SMC1, and FancD2 participate in the transient IR-induced S-phase arrest; [14][15][16][17] and Brca1 and hRad17 have been implicated in the G2/M checkpoint. 18,19 Previous research has shown that it is possible to correct A-T deficiencies by DNA transfection in cell culture, 20,21 and that overexpression of the normal ATM protein does not cause cellular abnormalities. 22 The large size of the ATM cDNA (B9 kb) required to correct the hereditary deficiency in A-T limits use of many vectors for gene delivery.…”