ORC binding, gene amplification, and the nature of metazoan replication origins.

Abstract

Genomic DNA replication is a critical process shaped by strong evolutionary pressures. Rapid replication can give a viral or cellular genome an immediate advantage over its competitors as long as requisitely high accuracy is not sacrificed. Prokaryotes employ an extremely fast, accurate DNA replication mechanism utilizing sophisticated multiprotein machines (Kornberg and Baker 1992; Baker and Bell 1998). Genome duplication is controlled by the assembly and activation of bidirectional replication forks at specific replication origins, under the control of initiator proteins that bind origins sequence specifically. For example, Escherichia coli oriC binds multiple copies of the initiator protein, dnaA, at repeated motifs. In an ATP-dependent manner dnaA then locally unwinds the DNA, facilitates helicase loading, and organizes the assembly of polymerases, primases, and other components of the replication fork. Special proteins are required to assemble and maintain this unwieldy machinery. Thus, loading the ring-shaped six-protein dnaB helicase around the DNA requires dnaC, whereas proteins such as tau probably act as scaffolds, organizing replication fork components (including polymerases). Despite the greater complexity of eukaryotic chromosomes, work carried out in Saccharomyces cerevisiae indicates that its genome replicates via a strongly similar mechanism (Leatherwood 1998). Each yeast chromosome contains multiple discrete replication origins where prereplication complexes are assembled during late M and G1 phases. Fully formed, bidirectional replication forks are then activated during S phase. Replication origins can be readily identified as autonomously replicating sequence (ARS) elements that support the propagation of extrachromosomal plasmids. Budding yeast origins are ∼100 bp in size and share an A/T-rich ARS consensus sequence (ACS) that is essential for origin function. Six proteins that form the origin recognition complex (ORC) bind to the ACS in an ATP-dependent manner throughout the cell cycle (Bell and Stillman 1992). Beginning in late mitosis, Cdc6p interacts with origin-bound ORC to bring the six-protein MCM complex, a putative replicative helicase, onto the DNA (Aparicio et al. 1997; Perkins and Diffley 1997; Tanaka et al. 1997). In a late step requiring cyclin-dependent kinase (cdk) activity, Cdc45p is added to the prereplication complex, possibly as a scaffold component (Zou and Stillman 1999). Activation of another origin-associated kinase, Cdc7p, by its regulatory subunit Dbf4p, probably serves as the final signal activating replication fork movement (Dowell et al. 1994; Hardy 1998; Brown and Kelly 1999). By the time prereplication complexes fire, high cdk activity precludes the assembly of new complexes. Because a checkpoint ensures that the cell will not divide until replication is complete, and high levels of cdk activity are not reversed until mitosis is nearly finished, the genome will be replicated once and only once per cell cycle.