Stepwise assembly of initiation proteins at budding yeast replication origins in vitro

The initiation of DNA replication in the budding yeast Saccharomyces cerevi siae occurs in two sequential and mutually exclusive steps. Prereplicative complexes (pre-RCs) containing origin recognition complex (ORC). Cdc6p, and the MCM2-7 proteins assemble only under conditions of low cyclin-dependent kinase (Cdk) activity during G(1), whereas origin activation is driven by the increase in Cdk activity at the end of G(1). As a first step toward the reconstitution of this two-step process in vitro, we describe a system in which extracts prepared from G(1)-arrested cells promote sequential assembl y of ORC, Cdc6p, and MCM2-7 proteins onto exogenously added origin-containi ng DNA. This reaction requires an intact ARS consensus sequence and require s ATP for two distinct steps. Extracts from cells arrested in mitosis also can support the binding of ORC but are unable to load either Cdc6p or MCM2- 7 proteins. This system should be useful for studying the mechanism and reg ulation of pre-RC assembly.