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The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA (rDNA) arrays. We have previously reported that in the absence of SIR2, a de-repressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler. more...

There are approximately 500 known origins of replication in the yeast genome, and the process by which DNA replication initiates at these locations is well understood. In particular, these sites are made competent to initiate replication by loading of the Mcm replicative helicase prior to the start of S phase; thus, ‘a site that binds Mcm in G1’ might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, we previously showed that known origins are typically bound by a single Mcm double hexamer, loaded adjacent to the ARS consensus sequence (ACS). Here, we extend this analysis from known origins to the entire genome, identifying candidate Mcm binding sites whose signal intensity varies over at least three orders of magnitude. Published data quantifying single-stranded DNA (ssDNA) during S phase revealed replication initiation among the most abundant 1600 of these sites, with replication activity decreasing with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5500 sites. Specifically, these sites: (1) appeared in intergenic nucleosome-free regions flanked on one or both sides by well-positioned nucleosomes; (2) were flanked by ACSs; and (3) exhibited a pattern of GC skew characteristic of replication initiation. We conclude that, if sites at which Mcm double hexamers are loaded can function as replication origins, then DNA replication origins are at least threefold more abundant than previously assumed, and we suggest that replication may occasionally initiate in essentially every intergenic region. These results shed light on recent reports that as many as 15% of replication events initiate outside of known origins, and this broader distribution of replication origins suggest that S phase in yeast may be less distinct from that in humans than widely assumed. more...