Abstract
The organization of DNA replication location and timing is regulated by the establishment of replication origins. Origins are established in G1 by the loading of a pair of MCMs, the catalytic core of the replicative helicase, as an inactive double‐hexamer complex. Activation of MCM during S phase is the rate‐limiting step for replication initiation, and therefore regulates where and when replication occurs. We have previously shown that origins at which multiple MCM complexes are loaded have a higher probability of initiation and therefore replicate earlier, on average. Bulk biochemical experiments suggest the ARS1, a early‐firing budding yeast origin, loads, on average, about three MCM complexes in G1, whereas ARS316, a late‐firing origin, loads less than one. However, these bulk measurements have two major drawbacks. First, the required normalization controls make our absolute stoichiometry estimates uncertain. Second, they only reveal the average numbers of MCM's loaded; they provide no insight into the distribution of MCM stoichiometry, information which could elucidate how MCM loading stoichiometry is controlled.