Timothy P. Heffernan, Ph.D.

Dissertation research performed under the direction of William K. Kaufmann

ABSTRACT
The S checkpoint is activated in response to diverse forms of DNA damage and slows the rate of DNA synthesis by transiently inhibiting the initiation of new replicons. Ionizing radiation (IR)-induced inhibition of replicon initiation is an active checkpoint response dependent on the transducer kinase ATM. ATM inhibits replicon initiation by stimulating the proteolysis of the Cdk2-activating phosphatase Cdc25A. Inhibition of replicon initiation is also observed in human cells exposed to low fluences of ultraviolet radiation (UV), however, the mechanism of inhibition is not fully understood. Studies were undertaken to test the hypothesis that UVC-induced inhibition of replicon initiation is an active S checkpoint response. To test this hypothesis, human cell lines containing inherited or engineered mutations in checkpoint genes were examined for their ability to inhibit replicon initiation in response to UVC. Velocity sedimentation of nascent DNA molecules revealed a ~50% inhibition of replicon initiation when normal fibroblasts were treated with a low fluence of UVC (1 J/m2). Fibroblasts from patients with ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and AT-like disorder (ATLD) responded with normal inhibition of replicon initiation when exposed to UVC. In contrast, human cells expressing a kinase dead ATR (AT- and Rad3-related) allele failed to inhibit replicon initiation following treatment with UVC. Inhibition of ATR function correlated with an attenuation of ATR-dependent Chk1 phosphorylation. Overexpression of a kinase-dead Chk1 allele abrogated the UVC-induced S checkpoint. ATM- and ATR-dependent S checkpoint responses were contrasted further by showing that human cells treated with low dose IR inhibited Cdk2/cyclin E activity by ~50% in association with the degradation of Cdc25A. Low dose UVC failed to induce inhibition of Cdk2/cyclin E or Cdc25A degradation. Overexpression of Dbf4 in human cells reversed the UVC-induced inhibition of replicon initiation, but had no effect on IR-induced inhibition of DNA synthesis. Dbf4 physically interacts with Chk1 inside cells, and is a substrate for Chk1-dependent phosphorylation in vitro; these findings implicate Cdc7/Dbf4 as a target of the UVC-induced S checkpoint. These data suggest that human cells have evolved multiple mechanisms to inhibit replicon initiation in response to DNA damage.