PMC:548366 / 34009-37664 JSONTXT

{"project":"2_test","denotations":[{"id":"15687385-11429609-77096054","span":{"begin":375,"end":377},"obj":"11429609"},{"id":"15687385-11486016-77096054","span":{"begin":375,"end":377},"obj":"11486016"},{"id":"15687385-9353247-77096054","span":{"begin":375,"end":377},"obj":"9353247"},{"id":"15687385-11125146-77096055","span":{"begin":756,"end":758},"obj":"11125146"},{"id":"15687385-11967157-77096056","span":{"begin":759,"end":761},"obj":"11967157"},{"id":"15687385-11532929-77096057","span":{"begin":959,"end":961},"obj":"11532929"},{"id":"15687385-11555648-77096058","span":{"begin":962,"end":964},"obj":"11555648"},{"id":"15687385-10995389-77096058","span":{"begin":962,"end":964},"obj":"10995389"},{"id":"15687385-11046155-77096058","span":{"begin":962,"end":964},"obj":"11046155"},{"id":"15687385-11737877-77096059","span":{"begin":1383,"end":1385},"obj":"11737877"},{"id":"15687385-11950940-77096060","span":{"begin":1386,"end":1388},"obj":"11950940"},{"id":"15687385-9702188-77096061","span":{"begin":1536,"end":1538},"obj":"9702188"},{"id":"15687385-10766248-77096062","span":{"begin":1708,"end":1710},"obj":"10766248"},{"id":"15687385-11836525-77096063","span":{"begin":1711,"end":1713},"obj":"11836525"},{"id":"15687385-12192004-77096064","span":{"begin":1714,"end":1716},"obj":"12192004"},{"id":"15687385-14672932-77096065","span":{"begin":1717,"end":1719},"obj":"14672932"},{"id":"15687385-14672932-77096066","span":{"begin":3003,"end":3005},"obj":"14672932"}],"text":"Probable significance of the strictly ordered binding of Cdc6 and Cdt1 to chromatin\nRe-licensing must be avoided in the normal cell cycle, and there seems to be a number of mechanisms to ensure this. First, the licensing reaction is thought to occur in the absence of Cdk activity on which other cell cycle-related events, such as DNA replication and mitosis, are dependent (42–44). This difference in dependency on Cdk activity temporally separates the licensing reaction from the other cell cycle processes. Second, there is an endogenous Cdt1 inhibitor protein, geminin, in higher eukaryotic cells. The expression and degradation of geminin seems strictly regulated in the cell cycle, and geminin biochemically prevents uncontrolled activation of Cdt1 (23,38). Third, the amounts of Cdc6 and Cdt1 in the cells seem strictly controlled in the cell cycle. The mechanism limits the emergence of the licensing reaction during a small window of the cell cycle (27,45–47). In this study, we presented evidence of an order to the chromatin-binding of Cdc6 and Cdt1 for these proteins to function in the licensing system. This mechanism may also provide another regulatory mechanism to prevent the re-licensing of DNA.\nIt has been reported that the binding of ATP to Cdc6 is required for its binding with chromatin, and the hydrolysis of the bound ATP is necessary for loading of Mcm2-7 (37,39). Since Cdc6 is a member of the AAA+ superfamily and exhibits sequence similarity to RF-C, it is presumed to function like a clamp loader protein (13). However, there is no evidence of direct interaction between Cdc6 and Mcm complex, though accumulative evidence suggests that Cdt1 interacts with Cdc6 and Mcm complex (19,21,30,40). Therefore, Cdt1 may play a role in the linking of Cdc6 and Mcm complex in this process. Given that Cdc6 is an ATPase, it is interesting to know whether the assembly of Cdt1 onto pre-RC requires Cdc6-dependent hydrolysis of ATP. For this purpose, we have observed that Cdt1 bound to chromatin in the presence of ATPγS, a non-hydrolyzable analog of ATP. Moreover, the licensing reaction and Mcm4 loading were also detected after isolation of the chromatin from the ATPγS-containing reaction mixture followed by an incubation with ATP and the Mcm fraction (T. Tsuyama et al., unpublished observation). These results suggest that ATP hydrolysis catalyzed by Cdc6 is not required for the functional loading of Cdt1.\nWhy does the licensing reaction require a strictly ordered chromatin-binding by Cdc6 and Cdt1? We have shown that Cdc6 loaded on a chromatin that had already associated with Cdt1 appeared to function in the licensing reaction with Mcm2-7 and Cdt1 supplied after the Cdc6-loading. The result suggests that the activity of Cdt1, not Cdc6, was affected when the order of the binding was reversed. Recently, Cook and co-workers have shown that Cdc6 binds to Cdt1 at its N-terminal non-catalytic domain, and this interaction promotes the association of Cdt1 and Mcm complex (40). It is possible that the interaction between Cdt1 and Cdc6 only occurs when Cdc6 is associated with ORC prior to the binding of Cdt1.\nIn this paper, we provide evidence that the DNA replication licensing system requires a strict order of chromatin binding of Cdc6 and Cdt1, in which Cdc6 must bind to chromatin prior to the association of Cdt1 for the licensing reaction to occur. The phenomenon is evidence for cooperation between Cdc6 and Cdt1 for the loading of Mcm2-7 onto chromatin during the licensing process. Elucidating the mechanism of cooperation between Cdc6 and Cdt1 would provide a clue to the molecular basis of the licensing system."}