CDK‐dependent assembly of replication proteins at the initiation step of chromosomal DNA replication

Abstract

Replication proteins assemble onto origins in two successive steps; formation of the pre‐RC in late M and G1 phases and assembly of other replication proteins including DNA polymerases to initiate DNA synthesis when CDK is activated from G1/S boundary. Sld2 and Sld3 are phosphorylated by CDK and bind to N‐terminal and C‐terminal tandem BRCT domains of Dpb11, respectively. Their interactions are essential and sufficient for the CDK‐dependent initiation of chromosomal DNA replication. However, how these interactions promote initiation of chromosomal DNA replication has not been elucidated.We have argued that the CDK‐dependent interaction between Sld2 and Dpb11 facilitates formation of the pre‐LC containing Dpb11, Sld2, Polε and GINS. The pre‐LC is a fragile complex that is detected only with the aid of a cross‐linking agent in vivo and is formed in a CDK‐dependent and pre‐RC‐independent manner. We can now reconstitute the pre‐LC from purified proteins. To recruit replication proteins, such as GINS and Polε, to origins, the pre‐LC may bind to phosphorylated Sld3, which associates with origins in a Cdc45‐dependent manner. Moreover, Sld3 seems to facilitate formation of an active replicative helicase, the CMG complex consisting of Cdc45, MCM and GINS. We, thus, propose that the interactions between Sld2, Sld3 and Dpb11 recruit replication proteins to origins and remodel them to form an active helicase at origins.