DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone chaperone Asf1 and MCM2-7, the putative replicative helicase, are connected through a histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and similar defects arise from overproduction of new histone H3-H4 that compromises Asf1 function. These data link Asf1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork progression and histone supply and demand.

Proper genome packaging requires coordination of both DNA and histone metabolism. While histone gene transcription and RNA processing adequately provide for scheduled needs, how histone supply adjusts to unexpected changes in demand remains unknown. Here, we reveal that the histone chaperone nuclear autoantigenic sperm protein (NASP) protects a reservoir of soluble histones H3-H4. The importance of NASP is revealed upon histone overload, engagement of the reservoir during acute replication stress, and perturbation of Asf1 activity. The reservoir can be fine-tuned, increasing or decreasing depending on the level of NASP. Our data suggest that NASP does so by balancing the activity of the heat shock proteins Hsc70 and Hsp90 to direct H3-H4 for degradation by chaperone-mediated autophagy. These insights into NASP function and the existence of a tunable reservoir in mammalian cells demonstrate that contingency is integrated into the histone supply chain to respond to unexpected changes in demand.