EVIDENCE FOR A CDC6P-INDEPENDENT MITOTIC RESETTING EVENT INVOLVING DNA-POLYMERASE-ALPHA

Eukaryotic DNA replication is limited to once per cell cycle because c yclin-dependent kinases (cdks), which are required to fire origins, al so prevent re-replication. Components of the replication apparatus, th erefore, are 'reset' by cdk inactivation at the end of mitosis, In bud ding yeast, assembly of Cdc6p-dependent prereplicative complexes (pre- RCs) at origins can only occur during G(1) because it is blocked by cd k1 (Cdc28) together with B cyclins (Clbs), Here we describe a second, separate process which is also blocked by Cdc28/Clb kinase and, theref ore, can only occur during G(1); the recruitment of DNA polymerase alp ha-primase (pol alpha) to chromatin, The recruitment of pol alpha to c hromatin during G(1) is independent of pre-RC formation since it can o ccur in the absence of Cdc6 protein. Paradoxically, overproduction of Cdc6p can drive both dephosphorylation and chromatin association of po l alpha. Overproduction of a mutant in which the N-terminus of Cdc6 ha s been deleted is unable to drive pol alpha chromatin binding. Since t his mutant is still competent for pre-RC formation and DNA replication , we suggest that Cdc6p overproduction resets pol alpha chromatin bind ing by a mechanism which is independent of that used in pre-RC assembl y.