Chromatin architecture sets origin licensing capacity
Replication origin licensing enables complete and faithful genome duplication, yet how this process is regulated by chromatin in vivo remains unclear. Using MCM-ChEC-seq to track helicase loading from metaphase through G1 in budding yeast, we find that licensing occurs in a rapid, synchronous burst at mitotic exit followed by an early plateau despite continued permissive cell-cycle conditions and persistent ORC binding at origins. Here we show that chromatin architecture at replication origins quantitatively constrains helicase loading, thereby limiting the extent of origin licensing. Histone H3K56 acetylation marks newly replicated chromatin and is removed at S-phase exit by the deacetylases Hst3 and Hst4. Persistent H3K56ac severely impairs MCM loading despite normal ORC occupancy, indicating that chromatin limits licensing at the helicase-loading step. Strikingly, deletion or catalytic inactivation of the chromatin remodeler Isw2 increases licensing by approximately 40 percent in wild-type cells and fully suppresses the licensing defect in hst3 hst4 mutants, identifying Isw2 as a physiological inhibitor of origin licensing. Isw2-dependent nucleosome repositioning narrows the origin nucleosome-depleted region and restricts helicase loading. Together, these findings demonstrate that chromatin architecture sets origin licensing capacity. Newly replicated chromatin transiently adopts an Isw2-dependent inhibitory configuration that restricts helicase loading, and post-replicative chromatin maturation relieves - but does not eliminate - this constraint. Thus, genome-wide licensing reflects the integration of chromatin-encoded limits on origin usage with CDK-dependent control of licensing timing.