| DNA damage, whether through endogenous or exogenous sources, represents a major threat to genomic integrity. For this reason, cells have evolved regulatory networks, called checkpoints, which halt cell cycle progression in the presence of damaged DNA. Checkpoint responses are mediated by the activity of two kinases, ataxia telangiectasia mutated (ATM), and ATM and Rad3 related (ATR). ATM signaling is activated by the presence of double strand breaks, while ATR is activated by various other forms of DNA damage, and chemical inhibitors of DNA synthesis. ATM and ATR phosphorylate numerous substrates to inhibit cell cycle progression. Through direct phosphorylation of cell cycle machinery, and through activation of their respective effector kinases (Chk1 for ATR, Cds1 for ATM), ATM and ATR have been shown to inhibit the entry into S phase, S phase progression, and the entry into mitosis.; Work in Saccharomyces cerevisiae has shown activation of the yeast homologues of ATR and Chk1 in response to the alkylating agent methyl methanesulfonate (MMS), dependent upon the entrance of the cell into S phase. The response of higher eukaryotes to MMS damage, however, had not previously been characterized. For this reason, we investigated the response to MMS damage of DNA in the Xenopus cell free system.; We demonstrate that MMS damage of DNA activates the ATR-Chk1 branch of the DNA damage checkpoint, dependent on attempted replication of the damaged DNA. We have also identified a novel, DNA damage checkpoint independent pathway to inhibit of DNA replication in response to damage, shown to act at the level of initiation of replication. We further demonstrate that destruction of the replication licensing factor Cdt1 is partly responsible for this inhibition, but that there is another, non-proteolytic pathway activated that also inhibits replication initiation. Finally, we have attempted to isolate this non-proteolytic activity by fractionation of Xenopus extract exposed to MMS damaged DNA. Together, the data presented here demonstrate the operation of both checkpoint dependent and independent mechanisms to halt cell cycle progression in response to MMS damage of DNA. |
