Evidence for a Cdc6p-independent mitotic resetting event involving DNA polymerase α

Eukaryotic DNA replication is limited to once per cell cycle because cyclin-dependent kinases (cdks), which are required to fire origins, also prevent re-replication. Components of the replication apparatus, therefore, are 'reset' by cdk inactivation at the end of mitosis, In budding yeast, assembly of Cdc6p-dependent prereplicative complexes (pre-RCs) at origins can only occur during G(1) because it is blocked by cdk1 (Cdc28) together with B cyclins (Clbs), Here we describe a second, separate process which is also blocked by Cdc28/Clb kinase and, therefore, can only occur during G(1); the recruitment of DNA polymerase alpha-primase (pol alpha) to chromatin, The recruitment of pol alpha to chromatin during G(1) is independent of pre-RC formation since it can occur in the absence of Cdc6 protein. Paradoxically, overproduction of Cdc6p can drive both dephosphorylation and chromatin association of pol alpha. Overproduction of a mutant in which the N-terminus of Cdc6 has been deleted is unable to drive pol alpha chromatin binding. Since this mutant is still competent for pre-RC formation and DNA replication, we suggest that Cdc6p overproduction resets pol alpha chromatin binding by a mechanism which is independent of that used in pre-RC assembly.