ASSOCIATION OF RPA WITH CHROMOSOMAL REPLICATION ORIGINS REQUIRES AN MCM PROTEIN, AND IS REGULATED BY RAD53, AND CYCLIN-DEPENDENT AND DBF4-DEPENDENT KINASES

Eukaryotic cells use multiple replication origins to replicate their l arge genomes, Some origins fire early during S phase whereas others fi re late. In Saccharomyces cerevisiae, initiator sequences (ARSs) are b ound by the origin recognition complex (ORC), Cdc6p synthesized at the end of mitosis joins ORC and facilitates recruitment of Mcm proteins, which renders origins competent to fire. However, origins fire only u pon the subsequent activation of S phase cyclin-dependent kinases (S-C DKs) and Dbf4/Cdc7 at the G(1)/S boundary. We have used a chromatin im munoprecipitation assay to measure the association with ARS sequences of DNA primase and the single-stranded DNA binding replication protein A (RPA) when fork movement is inhibited by hydroxyurea (HU), RPA's as sociation with origins requires S-CDKs, Dbf4/Cdc7 kinase and an Mcm pr otein. The recruitment of DNA primase depends on RPA. Furthermore, ear ly- and late-firing origins differ not in the timing of their recruitm ent of an Mcm protein, but in the timing of RPA's recruitment. RPA is recruited to early but not to late origins in HU. We also show that Ra d53 kinase is required to prevent RPA association with a late origin i n HU, Our data suggest that the origin unwinding accompanied by RPA as sociation is a key step, regulated by S-CDKs, Dbf4/Cdc7 and Rad53p. Th us, in the presence of active S-CDKs and Dbf4/Cdc7, Mcms may open orig ins and thereby facilitate the loading of RPA.