Phosphorylation of serines 635 and 645 of human Rad17 is cell cycle regulated and is required for G(1)/S checkpoint activation in response to DNA damage

ATR [ataxia-telangiectasia-mutated (ATM)- and Rad3-related] is a protein ki nase required for both DNA damage-induced cell cycle checkpoint responses a nd the DNA replication checkpoint that prevents mitosis before the completi on of DNA synthesis. Although ATM and ATR kinases share many substrates, th e different phenotypes of ATM- and ATR-deficient mice indicate that these k inases are not functionally redundant. Here we demonstrate that ATR but not ATM phosphorylates the human Rad17 (hRad17) checkpoint protein on Ser(635) and Ser(545) in vitro. In undamaged synchronized human cells, these two si tes were phosphorylated in late G(1), S, and G(2)/M, but not in early-mid G 1. Treatment of cells with genotoxic stress induced phosphorylation of hRad 17 in cells in early-mid G1. Expression of kinase-inactive ATR resulted in reduced phosphorylation of these residues, but these same serine residues w ere phosphorylated in ionizing radiation (IR)-treated ATM-deficient human c ell lines. IR-induced phosphorylation of hRad17 was also observed in ATM-de ficient tissues, but induction of Ser(645) was not optimal. Expression of a hRad17 mutant, with both serine residues changed to alanine, abolished IR- induced activation of the G(1)/S checkpoint in MCF-7 cells. These results s uggest ATR and hRad17 are essential components of a DNA damage response pat hway in mammalian cells.