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-