The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function. Initiation of DNA replication occurs at multiple genomic loci, termed origins of replication. In Saccharomyces cerevisiae, replication origins were originally identified as short (;150-base-pair [bp]) autonomously replicating sequence (ARS) elements that were sufficient for the maintenance of episomes. The origin recognition complex (ORC) binds the ARS consensus sequence (ACS), an 11-bp T-rich sequence that is necessary but not sufficient for origin activity. During G1, ORC coordinates the recruitment of several additional replication factors to load the replicative DNA helicase, the Mcm2-7 complex, onto origin DNA to form the prereplicative complex (pre-RC) (for review, see Sclafani and Holzen 2007).The S. cerevisiae genome contains, by various metrics, 6000-40,000 potential ACS sequence matches, of which only a few hundred are bound specifically by ORC. Although active transcription of a sequence has been shown to prevent ORC binding and pre-RC formation (Mori and Shirahige 2007), the large majority of potential ACS matches are intergenic, suggesting that additional chromosomal features are required to define the subset of these sites that are bound by ORC and act as replication origins.All cellular events involving genomic DNA must operate within their chromosomal context. Nucleosomes are the most basic elements of chromatin structure. Nearly 80% of S. cerevisiae DNA is incorporated into stable nucleosomes, and their position relative to regulatory elements is a critical component of gene regulation. Significant regions of the genome are not in complex with nucleosomes, and are referred to as nucleosome-free regions (NFRs). NFRs represent particularly accessible parts of the genome that are frequently the site of multiprotein assemblies that regulate or perform key DNA templated processes (for review, see Rando and Chang 2009).The DNA replication program has been shown to be regulated by the local chromatin environment (Donaldson 2005). Although progress has been made in establishing that chromatin modifications impact the activation of replication origins (Vogelauer et al. 2002;Knott et al. 2009), it has also been shown that nucleosome positioning is critical for origin function. Early nucleosome mapping experiments on a plasmid containing the ARS1 origi...

The dynein-related AAA ATPase Rea1 is a preribosomal factor that triggers an unknown maturation step in 60S subunit biogenesis. Using electron microscopy, we show that Rea1's motor domain is docked to the pre-60S particle and its tail-like structure, harboring a metal ion-dependent adhesion site (MIDAS), protrudes from the preribosome. Typically, integrins utilize a MIDAS to bind extracellular ligands, an interaction that is strengthened under applied tensile force. Likewise, the Rea1 MIDAS binds the preribosomal factor Rsa4, which is located on the pre-60S subunit at a site that is contacted by the flexible Rea1 tail. The MIDAS-Rsa4 interaction is essential for ATP-dependent dissociation of a group of non-ribosomal factors from the pre-60S particle. Thus, Rea1 aligns with its interacting partners on the preribosome to effect a necessary step on the path to the export-competent 60S subunit.

Summary Activation of the eukaryotic replicative DNA helicase, the Mcm2-7 complex, requires phosphorylation by Cdc7/Dbf4 (Dbf4-dependent kinase or DDK), which, in turn, depends on prior phosphorylation of Mcm2-7 by an unknown kinase(s). We identified DDK phosphorylation sites on Mcm4 and Mcm6 and found that phosphorylation of either subunit suffices for cell proliferation. Importantly, prior phosphorylation of either S/T-P or S/T-Q motifs on these subunits is required for DDK phosphorylation of Mcm2-7 and for normal S phase passage. Phosphomimetic mutations of DDK target sites bypass both DDK function and mutation of the priming phosphorylation sites. Mrc1 facilitates Mec1 phosphorylation of the S/T-Q motifs of chromatin-bound Mcm2-7 during S phase to activate replication. Genetic interactions between priming site mutations and MRC1 or TOF1 deletion support a role for these modifications in replication fork stability. These findings identify new mechanisms to modulate origin firing and replication fork assembly during cell cycle progression.