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The S checkpoint response to ultraviolet radiation (UVC) that inhibits replicon initiation is dependent on the ATR and Chk1 kinases. Downstream effectors of this response, however, are not well characterized. Data reported here eliminated Cdc25A degradation and inhibition of Cdk2-cyclin E as intrinsic components of the UVC-induced pathway of inhibition of replicon initiation in human cells. A sublethal dose of UVC (1 J/m(2)), which selectively inhibits replicon initiation by 50%, failed to reduce the amount of Cdc25A protein or decrease Cdk2-cyclin E kinase activity. Cdc25A degradation was observed after irradiation with cytotoxic fluences of UVC, suggesting that severe inhibition of DNA chain elongation and activation of the replication checkpoint might be responsible for the UVC-induced degradation of Cdc25A. Another proposed effector of the S checkpoint is the Cdc7-Dbf4 complex. Dbf4 interacted weakly with Chk1 in vivo but was recognized as a substrate for Chk1-dependent phosphorylation in vitro. FLAG-Dbf4 formed complexes with endogenous Cdc7, and this interaction was stable in UVC-irradiated HeLa cells. Overexpression of FLAG- or Myc-tagged Dbf4 abrogated the S checkpoint response to UVC but not ionizing radiation. These findings implicate a Dbf4-dependent kinase as a possible target of the ATR- and Chk1-dependent S checkpoint response to UVC.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by neuronal degeneration accompanied by ataxia, telangiectasias, acute cancer predisposition, and sensitivity to ionizing radiation (IR). Cells from individuals with AT show unusual sensitivity to IR, severely attenuated cell cycle checkpoint functions, and poor p53 induction in response to IR compared with normal human fibroblasts (NHFs). The gene mutated in AT (ATM) has been cloned, and its product, pATM, has IR-inducible kinase activity. The AT phenotype has been suggested to be a consequence, at least in part, of an inability to respond appropriately to oxidative damage. To test this hypothesis, we examined the ability of NHFs and AT dermal fibroblasts to respond to t-butyl hydroperoxide and IR treatment. AT fibroblasts exhibit, in comparison to NHFs, increased sensitivity to the toxicity of t-butyl hydroperoxide, as measured by colony-forming efficiency assays. Unlike NHFs, AT fibroblasts fail to show G(1) and G(2) phase checkpoint functions or to induce p53 in response to t-butyl hydroperoxide. Treatment of NHFs with t-butyl hydroperoxide activates pATM-associated kinase activity. Our results indicate that pATM is involved in responding to certain aspects of oxidative damage and in signaling this information to downstream effectors of the cell cycle checkpoint functions. Our data further suggest that some of the pathologies seen in AT could arise as a consequence of an inability to respond normally to oxidative damage.