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

Competition for MCM Loading at Origins Establishes Replication Timing Patterns [preprint]

Loading of the MCM replicative helicase onto origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The number of MCM loaded on origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide characteristics of MCM loading and their direct effect on replication timing remain unclear. In order to probe MCM loading dynamics and its effect on replication timing, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their initiation during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels not limiting for MCM loading. Our results support a model in which the loading activity of origins, controlled by their ability to recruit ORC and compete for MCM, determines the number of helicases loaded, which in turn affects replication timing

The capacity of origins to load MCM establishes replication timing patterns

Loading of the MCM replicative helicase at origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The stoichiometry of MCM loaded at origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide regulation of MCM loading stoichiometry and its direct effect on replication timing remain unclear. In order to investigate why some origins load more MCM than others, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their replication during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels are not limiting for MCM loading. Our results support a model in which the loading capacity of origins is the primary determinant of MCM stoichiometry in wild-type cells, but that stoichiometry is controlled by origins\u27 ability to recruit ORC and compete for MCM when MCM becomes limiting