The capacity of origins to load MCM establishes replication timing patterns

Abstract

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’ ability to recruit ORC and compete for MCM when MCM becomes limiting.

Replication Stress, Genomic Instability, and Replication Timing: A Complex Relationship

“…In addition, this study revealed that more MCMs were loaded at early rather than late origins, thus increasing the probability that these origins fire early in the S-phase. Very recently, the same laboratory demonstrated that a reduction in the global cellular pool of MCMs causes significant delays in RT [ 138 ]. This strongly suggests that the expression level of MCMs is an important factor in the regulation of the RT program.…”

The consequences of differential origin licensing dynamics in distinct chromatin environments

“…In many species, replication timing in S phase shows a general (though not exclusive) pattern of early euchromatin replication and late heterochromatin replication 71, 72 . Differences in the density of G1 MCM loading at different individual loci have been implicated in these S phase replication timing differences; more ORC or MCM loading in G1 correlates with early firing origins 17, 60, 73 . Early firing in S and early G1 origin licensing may both be consequences of general DNA accessibility in euchromatin.…”

“…The relative amounts of loaded MCM complexes and MCM loading factors, particularly the Origin Recognition Complex, have been implicated in establishing replication timing programs within S phase. Furthermore, some links between MCM loading efficiency and chromatin features or chromatin modifying enzymes are known [16][17][18][19] . The Sir2 histone deacetylase is required for normal MCM distribution among budding yeast origins 20 .…”

Cell cycle-dependent chromatin dynamics at replication origins

“…Each replication origin in the genome has an inherent efficiency which is thought to be determined, at least in part, by ORC binding, Mcm2-7 loading, the recruitment of activation factors, and the local chromatin environment [2,20,26,27,36,37]. We had previously used genome-wide chromatin occupancy profiling to identify ORC-dependent small fragment occupancy footprints at replication origins in two discrete phases of the cell cycle --G1 and G2 [20].…”

“…In addition, the inherent initiation efficiency [2] and the time of activation [3,24] of each origin are also thought to be regulated, in part, by the local chromatin environment and the levels of chromatin associated ORC [20,25] and Mcm2-7 [26,27].…”