Mechanism of pre‐RC‐dependent cohesin loading in Xenopus egg extracts

Cohesin is a multi‐subunit, ring‐shaped protein complex that holds sister chromatids together from the time of their synthesis in S phase until they are segregated in anaphase. In vertebrates, cohesins first bind to chromosomes as cells exit mitosis, but the mechanism is unknown. Concurrent with cohesin binding to chromosomes, pre‐replication complexes (pre‐RCs) are assembled at origins of DNA replication through the sequential loading of the initiation factors ORC, Cdc6, Cdt1, and MCM2‐7. In S phase, pre‐RCs are sequentially acted on by two protein kinases, Cdc7 and Cdk2, leading to origin unwinding, replisome assembly, and DNA replication. Importantly, experiments performed in yeast suggest that localization of cohesin to chromatin prior to DNA synthesis is essential for establishment of cohesion between sisters in S phase. We have investigated the mechanism of cohesin loading using Xenopus egg extracts. As seen in yeast, chromatin‐recruitment of Xenopus cohesin requires Scc2‐Scc4. Scc2‐Scc4 itself binds stably to chromatin, and this process is dependent on pre‐RC formation. In contrast, Cdk2 was not required for Scc2‐Scc4 or cohesin loading. These results support a model in which pre‐RCs recruit Scc2‐Scc4 to chromatin, which in turn stimulates loading of cohesin. So far, we have detected no direct interactions between Scc2‐Scc4 and components of the pre‐RC, leaving open the question of how Scc2‐Scc4 interacts with pre‐RCs. Intriguingly, the Scc2‐Scc4 and cohesin complexes are loaded onto chromatin during the same narrow time window as Cdc7 kinase (after pre‐RC formation and before initiation). We are now focusing on Cdc7 as a potential molecular link between pre‐RCs and the cohesin machinery.