Dynamics of Pre-replicative Complex Assembly

Abstract

The pre-replicative complex (pre-RC) is formed at all potential origins of replication through the action of the origin recognition complex (ORC), Cdc6, Cdt1, and the Mcm2-7 complex. The end result of pre-RC formation is the loading of the Mcm2-7 replicative helicase onto origin DNA. We examined pre-RC formation in vitro and found that it proceeds through separable binding events. Origin-bound ORC recruits Cdc6, and this ternary complex then promotes helicase loading in the presence of a pre-formed Mcm2-7-Cdt1 complex. Using a stepwise pre-RC assembly assay, we investigated the fate of pre-RC components during later stages of the reaction. We determined that helicase loading is accompanied by dissociation of ORC, Cdc6, and Cdt1 from origin DNA. This dissociation requires ATP hydrolysis at a late stage of pre-RC assembly. Our results indicate that pre-RC formation is a dynamic process.Eukaryotic DNA replication initiates at origins of replication throughout the genome. The first event in this tightly regulated process is origin selection. In the budding yeast Saccharomyces cerevisiae, specific nucleotide sequences mark sites where replication can initiate. These sites are bound by the origin recognition complex (ORC) 3 (1, 2). As cells enter G 1 , ORC recruits Cdc6, Cdt1, and the Mcm2-7 replicative helicase to the origin DNA resulting in pre-replicative complex (pre-RC) formation (3). Activation of S phase kinases leads to the recruitment of numerous additional factors to the Mcm2-7 complex resulting in the formation of a pair of bi-directional replisomes (4). Multiple mechanisms regulate pre-RC formation to ensure that each origin can only initiate once per cell cycle (5). Thus, the pre-RC marks all potential sites of replication initiation in G 1 , forms the foundation for the assembly of the replisome, and is tightly regulated to ensure complete genome replication.Studies of S. cerevisiae DNA replication initiation have been greatly facilitated by the short (80 -120 bp), well defined origins of replication of this organism (6). These sequences include a conserved 11-bp autonomous replicating sequence consensus sequence and multiple 10 -15-bp B-elements. Much of our understanding of the pre-RC has come from in vitro origin binding assays. It was shown that the ORC-origin interaction is ATP-dependent (7) and that Cdc6 cooperatively binds the ORC-DNA complex (8). This ternary complex is also regulated by Cdc6 ATPase activity (9). In vitro pre-RC assembly in budding yeast extracts indicates sequential association of ORC, Cdc6, Cdt1, and Mcm2-7 with the origin (10, 11), and ATP hydrolysis by Cdc6 is required for .In this study, we examine pre-RC formation in vitro and find that the process can be separated into three distinct sequential binding events. The final step of Mcm2-7 complex loading correlates with reduced origin association of ORC, Cdc6, and Cdt1. The dissociation of these proteins from the origin requires ATP hydrolysis at a late step in pre-RC assembly. Our findings indicate that the pre-RC proteins as...

Cdt1p, through its interaction with Mcm6p, is required for the formation, nuclear accumulation and chromatin loading of the MCM complex

“…It was also shown that, without cooverexpression of Cdt1p, most of the overexpressed MCM subunits in yeast cell extracts formed sub-complexes other than the Mcm2-7p hexamer (Tsakraklides and Bell, 2010). These observations are consistent with our results that non-chromatinbound MCM proteins cannot form a heterohexameric complex after Cdt1p depletion in vivo.…”

“…It was reported that approximately equimolar Cdt1p was readily co-purified with the Mcm2-7p complex from yeast cell extracts (Kawasaki et al, 2006;Remus et al, 2009;Tsakraklides and Bell, 2010). It was also shown that, without cooverexpression of Cdt1p, most of the overexpressed MCM subunits in yeast cell extracts formed sub-complexes other than the Mcm2-7p hexamer (Tsakraklides and Bell, 2010).…”

Helicase Loading at Chromosomal Origins of Replication

“…In vitro studies show that after loading, Mcm2 -7 no longer requires ORC, Cdc6, or Cdt1 to associate with origin DNA (Donovan et al 1997;Randell et al 2006). Consistent with this observation, in vitro studies show that the helicase loading proteins are released from the origin upon Mcm2 -7 loading (Randell et al 2006;Tsakraklides and Bell 2010). In contrast to these findings, in vivo inactivation of S. cerevisiae ORC or Cdc6 in late G 1 (after Mcm2 -7 loading) results in loss of Mcm2 -7originassociation(Aparicioetal.1997;Semple et al 2006;Chen et al 2007).…”

“…ORC ATP hydrolysis is also essential but functions after Cdc6 hydrolysis and appears to regulate repeated Mcm2 -7 loading (Bowers et al 2004;Randell et al 2006). One likely role for ORC ATP hydrolysis is to cause the release of the other helicase loading factors from the origin (Tsakraklides and Bell 2010). Evidence from studies of S. cerevisiae suggests that a second consequence of ATP hydrolysis is to disable ORC DNA binding until it is able to bind another molecule of ATP.…”

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

“…Chromatinbound ORC-Cdc6 function to recruit a Cdt1- heptamer to replication origins (Fig. 1A, step 2; Speck et al 2005;Randell et al 2006;Speck and Stillman 2007;Tsakraklides and Bell 2010;Evrin et al 2013;Frigola et al 2013;Sun et al 2013). Using an in vitro Mcm2-7 loading system that employs a purified ORC, Cdc6, Cdt1, and Mcm2-7 hexamer and an origin-containing plasmid, we recently observed formation of a loading intermediate termed OCCM in the presence of a slowly hydrolysable ATP analog, ATP-gS, which captured a complex containing ORC-Cdc6 bound to a single Mcm2-7 hexamer and one to two copies of Cdt1 (Takara and Bell 2011;Evrin et al 2013;Sun et al 2013).…”