Regulation of Cellular and SV40 Virus Origins of Replication by Chk1-dependent Intrinsic and UVC Radiation-induced Checkpoints

Abstract

DNA replication is inhibited by DNA damage through cis effects on replication fork progression and trans effects associated with checkpoints. In this study, we employed a combined pulse labeling and neutral-neutral two-dimensional gel-based approach to compare the effects of a DNA damaging agent frequently employed to invoke checkpoints, UVC radiation, on the replication of cellular and simian virus 40 (SV40) chromosomes in intact cells. UVC radiation induced similar inhibitory effects on the initiation and elongation phases of cellular and SV40 DNA replication. The initiation-inhibitory effects occurred independently of p53 and were abrogated by the ATM and ATR kinase inhibitor caffeine, or the Chk1 kinase inhibitor UCN-01. Inhibition of cellular origins was also abrogated by the expression of a dominant-negative Chk1 mutant. These results indicate that UVC induces a Chk1- and ATR or ATM-dependent checkpoint that targets both cellular and SV40 viral replication origins. Loss of Chk1 and ATR or ATM function also stimulated initiation of cellular and viral DNA replication in the absence of UVC radiation, revealing the existence of a novel intrinsic checkpoint that targets both cellular and SV40 viral origins of replication in the absence of DNA damage or stalled DNA replication forks. This checkpoint inhibits the replication in early S phase cells of a region of the repetitive rDNA locus that replicates in late S phase. The ability to detect these checkpoints using the well characterized SV40 model system should facilitate analysis of the molecular basis for these effects.