Abstract
We have studied the rate of DNA synthesis, cell cycle distribution, formation of γ‐H2AX, and Rad51 nuclear foci and association of Rad51 with the nuclear matrix after treatment of HeLa cells with the interstrand crosslinking agent mitomycin C (MMC) in the presence of the kinase inhibitors caffeine and wortmannin. The results showed that MMC treatment arrested the cells in S‐phase and induced the appearance of γ‐H2AX and Rad51 nuclear foci, accompanied with a sequestering of Rad51 to the nuclear matrix. These effects were abrogated by caffeine, which inhibits the Ataxia‐telangiectasia mutated (ATM) and ATM‐ and Rad3‐related (ATR) kinases. However, wortmannin at a concentration that inhibits ATM, but not ATR did not affect cell cycle progression, damage‐induced phosphorylation of H2AX and Rad51 foci formation, and association with the nuclear matrix, suggesting that the S‐phase arrest induced by MMC is ATR‐dependent. These findings were confirmed by experiments with ATR‐deficient and AT cells. They indicate that the DNA damage ATR‐dependent S‐phase checkpoint pathway may regulate the spatiotemporal organization of the process of repair of interstrand crosslinks. J. Cell. Physiol. 211: 468–476, 2007. © 2006 Wiley‐Liss, Inc.