Identification of 1600 replication origins in S. cerevisiae

Reviewer #1 (Public Review):

The authors have previously employed micrococcal nuclease tethered to various Mcm subunits to the cut DNA to which the Mcm2-7 double hexamers (DH) bind. Using this assay, they found that Mcm2-7 DH are located on many more sites in the S. cerevisiae genome than previously shown. They then demonstrated that these sites have characteristics consistent with origins of DNA replication, including the presence of ARS consensus sequences, location of very inefficient sites of initiation of DNA replication in vivo, are free of nucleosomes, they contain a G-C skew and they locate to intergenic regions of the genome. The authors suggest, consistent with published single molecule results, that there are many more potential origins in the S. cerevisiae genome than previously annotated.

The results are convincing and are consistent with prior observations. The analysis of the origin associated features is informative.

Reviewer #2 (Public Review):

By mapping the sites of the Mcm2-7 replicative helicase loading across the budding yeast genome using high-resolution chromatin endogenous cleavage or ChEC, Bedalov and colleagues find that these markers for origins of DNA replication are much more broadly distributed than previously appreciated. Interestingly, this is consistent with early reconstituted biochemical studies that showed that the ACS was not essential for helicase loading in vitro (e.g. Remus et al., 2009, PMID: 19896182). To accomplish this, they combined the results of 12 independent assays to gain exceptionally deep coverage of Mcm2-7 binding sites. By comparing these sites to previous studies mapping ssDNA generated during replication initiation, they provide evidence that at least a fraction of the 1600 most robustly Mcm2-7-bound sequences act as origins. A weakness of the paper is that the group-based (as opposed to analyzing individual Mcm2-7 binding sites) nature of the analysis prevents the authors from concluding that all of the 1,600 sites mentioned in the title act as origins. The authors also show that the location of Mcm2-7 location after loading are highly similar in the top 500 binding sites, although the mobile nature of loaded Mcm2-7 double hexamers prevents any conclusions about the location of initial loading. Interestingly, by comparing subsets of the Mcm2-7 binding sites, they find that there is a propensity of at least a subset of these sites to be nucleosome depleted, to overlap with at least a partial match to the ACS sequence (found at all of the most well-characterized budding yeast origins), and a GC-skew. Each of which is a characteristic of previously characterized origins of replication.

Overall, this manuscript greatly broadens the number of sites that are capable of loading Mcm2-7 in budding yeast cells and shows that a subset of these additional sites act as replication origins. Although these sites do have a propensity to include a match to the ACS, these studies suggest that the mechanism of helicase loading in yeast and multicellular organisms is more similar than previously thought.