No abstract

DNA replication-coupled (RC) nucleosome assembly is mediated by histone chaperones and is fundamental for epigenetic inheritance and maintenance of genomic integrity. The mechanisms that promote this process are only partially understood. Here, we show that the histone chaperone FACT (facilitates chromatin transactions), consisting of Spt16 and Pob3, promotes newly synthesized histone H3-H4 deposition. We describe an allele of Spt16 (spt16-m) that has a defect in binding to H3-H4 and impairs their deposition onto DNA. Consistent with a direct role for FACT in RC nucleosome assembly, spt16-m displays synthetic defects with other histone chaperones associated with this process, CAF-1 and Rtt106. Importantly, we show that FACT physically associates with Rtt106 and that the acetylation of H3K56, a mark on newly synthesized H3, modulates this interaction. Therefore, FACT collaborates with CAF-1 and Rtt106 in RC nucleosome assembly.

DNA replication-coupled nucleosome assembly is essential to maintain genome integrity and retain epigenetic information. Multiple involved histone chaperones have been identified, but how nucleosome assembly is coupled to DNA replication remains elusive. Here we show that replication protein A (RPA), an essential replisome component that binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we propose that RPA functions as a platform for targeting histone deposition to replication fork, through which RPA couples nucleosome assembly with ongoing DNA replication.