Modulating MCM Levels Causes Differential Loading at Origins of Replication and Changes Replication Timing

DNA replication is a highly complex part of cell metabolism that ensures safe propagation of the genome through tight regulation of the expression, localization, and activity of a large number of factors. Replication starts from distinct sites in the genome and initiation events are temporally ordered in a manner that is, on average, highly reproducible across cell populations. The specific order with which different parts of the genome are replicated has been proposed to be important to processes such as gene expression, cell differentiation, development, and genome evolution. Nevertheless, the fundamental mechanisms that are responsible for establishing these timing programs remain elusive. Unlike in higher eukaryotes, DNA replication in budding yeast initiates at sequence-specific loci called origins of replication. The timing of initiation at these loci is determined by the activation of the main replicative helicase Minichromosome Maintenance (MCM) complex. Recent results have placed MCM in a key role in establishing a replication timing program that is reproducible but arises from stochastic activation of origins, as has been observed in yeast and higher eukaryotes. One particular model posits that the loading of multiple MCMs at individual origins increases the chances that origins will be activated earlier in S phase by a limited amount of initiation factors. To further test this model, we set out to examine the consequences of modulating MCM levels in budding yeast in order to ascertain their effects on the dynamics of helicase loading during G1 and subsequent replication timing. Overexpression of MCM2-7 had no effects on cell viability, cell cycle progression, MCM abundance at origins, or replication timing. On the other hand, depletion of Mcm4, one of the six obligate components of the MCM helicase, caused reduced viability, slower progression through S phase, and increased sensitivity to replication stress. Importantly, Mcm4 depletion led to differential reduction in MCM loading at origins during G1, with low MCM origins being disproportionately affected by reduced MCM pools. Finally, reduced MCM loading at origins of replication led to delayed replication during S phase. Our data support a model where the loading activity of origins, controlled by their ability to recruit ORC and compete for MCMs, determines the number of helicases loaded, which in turn has strong implications for replication timing.