Chromosomal Mcm2-7 distribution is the primary driver of the genome replication program in species from yeast to humans

Abstract

The spatio-temporal program of genome replication across eukaryotes is thought to be driven both by the uneven loading of pre-replication complexes (pre-RCs) across the genome at the onset of S-phase, and by differences in the timing of activation of these complexes during Sphase. To determine the degree to which distribution of pre-RC loading alone could account for chromosomal replication patterns, we identified the binding sites of the Mcm2-7 helicase complex, a key component of the pre-RC that is required for initiation of DNA replication, in budding yeast, fission yeast and mouse. In budding yeast, we detected Mcm2 binding in sharply focused peaks, generally with a single double hexamer bound at known origins of replication. In fission yeast, Mcm2 binding, while still concentrated at known origins, was more diffuse, often with 6 to 8 helicase complexes distributed along 0.5-1.5 kb sized origins, and with significantly more binding between origins. Finally, in mouse, we found even more diffuse Mcm2-7 distribution, with the density of Mcm2-7 binding in G1 recapitulating to a remarkable degree the replication program implemented in S-phase. Computer simulations that assign each licensed origin an equal probability of firing show that the observed Mcm2-7 density distribution in G1 across all three species largely recapitulated the DNA replication program. We conclude that the pattern of origin licensing from yeast to mammals is sufficient to explain most differences in replication timing without invoking an overarching temporal program of origin firing. ResultsInitiation of DNA replication requires activation of the MCM2-7 helicase complex (Mcm2-7) at sites at which this complex was loaded during the preceding G2/M and G1 phases [1][2][3][4].However, the order in which different regions of the genome complete replication is widely assumed to reflect not only the locations of Mcm2-7 loading but also a program of control above the level of loading, in which certain complexes are activated before others [5,6]. The distribution of Mcm2-7 is therefore expected to constrain but not fully determine the program of genome replication. Mcm2-7 encircles approximately 60 base pairs (bp) of DNA as a double hexamer whose monomer components are juxtaposed head-to-head at their N-termini with C-

Competition for MCM Loading at Origins Establishes Replication Timing Patterns

“…Our combination of MCM ChIP-seq and replication timing data also allow us to directly confirm in a single experiment previous conclusions, drawn from comparing data collected in different studies, that replication timing correlates with levels of MCM loading (Das et al, 2015;Foss et al, 2020). Although the correlation between MCM levels and replication timing is robust (Figure 4c, p > 10 -5 ), it is clearly not the only determining factor (r = 0.29-0.52).…”

“…Alternatively, in a multiple-MCM scenario, more than one MCM double-hexamer complex can be loaded at a single origin, so MCM occupancy can range from 0 to many. Both single-and multiple-MCM scenarios have been proposed (Das et al, 2015;Foss et al, 2020;de Moura et al, 2010;Das and Rhind, 2016;Yang et al, 2010).…”

“…Although excess loading of MCM is important for a successful S phase in response to replication stress, it has also been implicated in contributing to the replication timing program under optimal conditions. Genome-wide ChIP experiments in budding yeast have shown that MCM is loaded at origins in different amounts, and that the level of MCM loading correlates with the time in S phases at which an origin fires (Das et al, 2015;Foss et al, 2020). In addition, mutation of the B2 element in ARS1, which that has been shown to be important for MCM loading at that origin, causes reduced MCM loading and a delay in replication timing (Zou and Stillman, 2000;Das et al, 2015).…”

The capacity of origins to load MCM establishes replication timing patterns

“…This data points to a model where the more MCMs that are available for activation at an origin in S phase, the higher the likelihood that the specific origin will initiate early. Indeed, this MCM-stoichiometry model fits well with kinetic modeling of replication, which predicts that stochastic firing arising from differences in the availability of MCM in the presence of limiting initiation factors can give rise to the observed replication kinetics seen in budding yeast cells [ 21 , 22 , 26 ]. It is important to note that the MCM-stoichiometry models are equally valid whether MCM stoichiometry varies from 0 to 1 or 0 to many.…”

“…Although excess loading of MCM is important for a successful S phase under conditions of replication stress, it has also been implicated in contributing to the replication timing program under normal conditions. Genome-wide MCM mapping experiments in budding yeast have shown that MCM is loaded at origins in different amounts, and that the level of MCM loading correlates with the time in S phases at which an origin fires [ 20 , 21 ]. In addition, mutation of the B2 element in ARS1, which has been shown to be important for MCM loading at that origin, causes reduced MCM loading and a delay in replication timing [ 20 , 54 ].…”