Mechanisms that prevent DNA re-replication in the yeast Saccharomyces cerevisiae

Abstract

Every time a cell divides it must faithfully duplicate its genome before the cell divides. If replication initiates a second time (re-replication) before cytokinesis, cells can accumulate extensive DNA damage, which results in genomic instability, a hallmark of tumorigenesis. To prevent re-replication eukaryotic cells must inhibit the re-initiation of replication start sites, or origins, across the genome. Examples of both Cyclin-Dependent Kinase (CDK)-dependent and CDK-independent mechanisms have been identified that regulate the components of the pre-Replicative Complex (pre-RC) to prevent re-replication. The pre-RC is a multi-protein complex that assembles at origins during G1, before DNA replication begins. After an origin initiates pre-RC components must be prevented from reassembling at origins until the next cell cycle. When the mechanisms preventing re-replication in the yeast Saccharomyces cerevisiae are disrupted, unregulated replication occurs. Not all origins are capable of re-initiating during this re-replication. Rather, a subset of all potential origin sequences reform pre-RCs, and of those, only a portion re-initiates. The origins that re-initiate do not correlate with any other known subclass of origins (e.g. - early/late initiating origins).

(cont.) The inability of some origins to form pre-RCs during re-replication might be due to restrictive chromatin structure preventing pre-RC components from associating with origin DNA. Similarly, origins that form pre-RCs but do not re-initiate might be prevented from recruiting replication machinery due to a restrictive chromatin structure. In addition, these origins might not re-initiate because replication factors that function downstream of pre-RC components also could be regulated to prevent re-replication. One of the mechanisms that S. cerevisiae and other eukaryotes use to prevent re-replication is phosphorylating one or multiple subunits of the Origin Recognition Complex (ORC). In S. cerevisiae, Orc2 and Orc6 are both phosphorylated but have distinct mechanisms for preventing re-replication. Phosphorylating Orc2 results in the direct inhibition of pre-RC assembly whereas phosphorylating Orc6 helps stabilize CDK at origins. By contrast, of CDK helps to prevent re-replication, most likely through a combination of catalytic activity and steric hindrance.