FACT–(H3-H4) complex stimulates Pol α activity to coordinate DNA synthesis with nucleosome assembly

Abstract

DNA replication is an exquisitely regulated process that requires tight coordination at the eukaryotic replication fork, operating within the compacted chromatin environment. Minimizing exposure of naked DNA is critical to prevent nuclease degradation and genome instability, a hallmark of cancer. Cells, therefore, require a surveillance mechanism that links nucleosome assembly status to DNA replication. Here, we show that histone H3-H4 delivered by the chaperone facilitates chromatin transaction (FACT) directly stimulates the DNA polymerase activity of the polymerase α (Pol α, primase-polymerase complex) at replication forks. Histone H3-H4, but not histone H2A-H2B, promotes assembly of a FACT–(H3-H4)–Pol1 complex in S phase, where FACT associates with the Pol1 catalytic subunit in an H3-H4–dependent manner. This H3-H4–bridged interaction enhances Pol α primer extension on both DNA- and RNA-primed templates, increasing synthesis rate without altering primer patterning. Mechanistically, the Pol1 N-terminal domain, particularly its α1 and α2 helices, mediates complex assembly; the Pol1-Δα1α2 mutation disrupts this interaction and impairs primer extension in vitro. In cells, disrupting this complex reduces Okazaki fragment synthesis and slows replication fork progression. Among replication-coupled histone chaperones, only FACT interacts with Pol α, and this association is attenuated when chromatin assembly factor 1 (CAF-1)– and regulator of Ty1 transposition protein 106 (Rtt106)–dependent nucleosome assembly is compromised. Together, our results support a model whereby the FACT–(H3-H4)–Pol α complex serves as a sensor of H3-H4 availability, coordinating DNA synthesis with replication-coupled nucleosome assembly to safeguard genome stability.