The 3 Rs of Cdc6: Recruitment, Regulation, and Replication

Abstract

What is it? The cell division cycle genes CDC6 and CDC18 encode budding and fission yeast orthologs identified in a series of elegant screens for regulators of cell cycle progression performed independently by Leland Hartwell and Paul Nurse in the 1970s. In the subsequent three decades, orthologs have been found throughout Eukaryota and have been revealed to play key roles in two fundamental aspects of replication, recruiting replication components to chromosomes, and helping safeguard against multiple rounds of DNA replication.How does it do both? Cdc6 is part of a macro-molecular machine that assembles on chromatin to license the DNA for a single round of replication. Cdc6 collaborates with the six-subunit origin recognition complex (ORC) and Cdt1 to recruit the minichromosome maintenance (MCM) family of proteins to DNA, thereby forming the prereplication complex (see Figure 1Figure 1). Once the DNA has been licensed, S phase promoting factor, which consists of various regulatory kinases including Cdc7 and cyclin-dependent kinases (CDKs), triggers the prereplication complexes to fire. In licensing the DNA, Cdc6 simultaneously lays the groundwork for ensuring each segment of the chromosomal DNA is replicated only once each cell cycle: concomitant with origin firing, Cdc6 disassembles from the prereplication complex, thereby blocking rereplication. Cdc6 is thus part of a unidirectional switch at replication origins that is triggered only once per S phase.Figure 1Initiation of eukaryotic DNA replication. During G1 phase, ORC (composed of six subunits), Cdc6 (which likely acts as a multimer), Cdt1, and MCMs (a ring-shaped clamp composed of six subunits) assemble on chromosomes to form the prereplication complex. During S phase, CDK phosphorylates Cdc6, which in turn is either degraded by SCF (yeast) or relocalized to the cytoplasm (metazoans).View Large Image | View Hi-Res Image | Download PowerPoint SlideIs Cdc6 a clamp loader? A clamp loader is a protein complex that uses ATP binding and hydrolysis as molecular switches to load a ring-shaped DNA processivity factor, the clamp, onto DNA. Biochemical analyses of eukaryotic Cdc6 suggest that this is just how Cdc6 works. That is, Cdc6 likely acts as a multimer in which ATP binding and hydrolysis induce conformational changes that result in the recruitment of MCM – a putative ring-shaped clamp – to DNA (see Figure 1Figure 1). Despite having limited sequence similarity, the three-dimensional structures of clamp loaders are remarkably similar among prokaryotic, archaeal and eukaryotic organisms, and indeed, an archaeal Cdc6 ortholog has recently been shown to contain clamp loader structural elements.How is Cdc6 regulated? In addition to changes in activity via interaction with ATP, Cdc6 is regulated by changes in its abundance and localization. Yeast and metazoans regulate Cdc6 through CDK-mediated phosphorylation during early S phase. In yeast, phosphorylated Cdc6 is subsequently degraded in a Skp1, Cdc53/Cullin, F box receptor (SCF)-dependent ubiquitin-mediated pathway. In contrast, phosphorylation of mammalian Cdc6 promotes its export from the nucleus into the cytoplasm where it is ubiquitinated by the anaphase-promoting complex (APC) and degraded during mitosis (see Figure 1Figure 1). In fission yeast, overexpression of Cdc18 causes endoreduplication without an intervening mitosis. However, similar mutations that override the degradation or localization controls of Cdc6 in either budding yeast or metazoans are not sufficient to overcome the block to rereplication. In fact, recent studies in budding yeast indicate that regulation of Cdc6 is one of several redundant mechanisms that prevent rereplication in eukaryotic cells.