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A new MCM modification cycle regulates DNA replication initiation

ABSTRACT: The hexameric DNA helicase MCM (Mcm2-7) is a central regulatory target in eukaryotic replication. Chromatin-bound MCM is kept inactive during G1 phase and subsequently activated in S phase to initiate replication. During this transition, the only known chemical change on the Mcm2-7 proteins is the gain of multi-site phosphorylation that promotes recruitment of co-factors. As replication initiation is tied intimately to multiple biological cues, additional changes on these proteins can provide a second regulatory point. Here we describe a new MCM modification cycle mediated by SUMO that enables a negative regulation of replication initiation. We show that all MCM subunits undergo sumoylation upon loading at origins in G1 phase prior to MCM phosphorylation. Then bulk MCM sumoylation is lost as MCM phosphorylation rises. The pattern of MCM sumoylation suggests a negative role in replication. Indeed, increasing MCM sumoylation delays genome-wide replication initiation. Mechanistically, this is partly due to enhancing the recruitment of a conserved phosphatase that delays MCM phosphorylation and activation. By revealing a new MCM modification cycle and its role in replication, our findings suggest a new model, in which MCM sumoylation counterbalances kinase-based regulation to ensure accurate control of replication initiation.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE70407 | GEO | 2016/02/01

SECONDARY ACCESSION(S): PRJNA288566

REPOSITORIES: GEO

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Project description: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 to which MCM is bound in G1" might be considered to provide an operational definition of a replication origin. By fusing a subunit of Mcm to micrococcal nuclease, a technique referred to as "Chromatin Endogenous Cleavage", 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 3 orders of magnitude. Published data quantifying the production of ssDNA during S phase showed clear evidence of replication initiation among the most abundant 1600 of these sites, with replication activity decreasing in concert with Mcm abundance and disappearing at the limit of detection of ssDNA. Three other hallmarks of replication origins were apparent among the most abundant 5,500 sites. Specifically, these sites (1) appeared in intergenic nucleosome-free regions that were 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. Furthermore, the high resolution of this technique allowed us to demonstrate a strong bias for detecting Mcm double-hexamers downstream rather than upstream of the ACS, which is consistent with the directionality of Mcm loading by Orc that has been observed in vitro. We conclude that DNA replication origins are at least 3-fold 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 is widely assumed.

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A new MCM modification cycle regulates DNA replication initiation

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