A34
Significance of regulating Cdc6 during the initiation of DNA replication
Lena R. Kundu, Yuji Kumata, Takashi Tsuyama, Masayuki Seki, Takemi Enomoto, Shusuke Tada
Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan
Background
Genome instability, which could be brought about by the disruption of normal replication mechanism, severely affects proper cell functioning. Therefore, initiation of DNA replication must be tightly regulated to ensure stable maintenance of the genome. This is achieved at first by the sequential binding of ORC, Cdc6, Cdt1 and Mcm2-7 and thereby licensing the origins for DNA replication. Overexpression of Cdc6 and Cdt1 has been reported in a number of human cancer cell lines and the accurate execution of DNA replication is suggested to require a strict control of these proteins. However, the exact consequence of deregulating the licensing activities during DNA replication is yet to be explored.
Results and discussion
In this study, we focused on Cdc6 to elucidate the significance of its regulation during the course of DNA replication. To address this issue, we added recombinant Cdc6 to Xenopus egg extracts in order to create a state in which Cdc6 remained highly bound to chromatin even after origins had been licensed. The data support the surprising finding that deregulated Cdc6 actually inhibited DNA replication rather than inducing over-replication. The inhibition by Cdc6 occurred at a step after pre-RC formation and before Cdk2-dependent events independent of the activation of ATM/ATR-dependent checkpoint pathways. Detailed analyses suggest that excess Cdc6 suppresses the hyperphosphorylation of Mcm4 by Cdc7, as well as phosphorylation of Cdc7 itself on chromatin. Our results propose a possible regulatory mechanism of the initiation of DNA replication signifying the importance of a strict regulation of Cdc6 function during DNA replication.
