biostudies-literatureRouillon CNovo Nordisk FoundationIndependent Research Fund DenmarkEuropean Union Horizon 2020Lundbeck FoundationOncode InstituteJapanese Biochemical SocietyEuropean Research CouncilMarie Curie Individual FellowshipEuropean UnionDutch Research Council (NWO)Dutch Cancer SocietyNovo Nordisk Foundation Center for Protein ResearchNovo Nordisk FondenMarie Curie3770-3792https://www.ebi.ac.uk/biostudies/studies/S-EPMC10164577biostudies-literatureUnknown51(8)During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA polyNucleic acids researchCAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands.PMC10164577NNF21OC00674252014-6649[R198-2015-269ERC CoG 724436724436NNF14OC0012839851564789267741.018.201PI Anja Groth714.017.002NNF14CC0001101003210R313-2019-44884162084162ERC-StG 8515647016-00042BEckhardt BVBiran ARouillon CVerkennis AEEKollenstart LGroth AAlbanese PDelvaux de Fenffe CMde Laat Wvan Laar TKnipscheer PKrijger PHLRicci GDekker NHMattiroli FGruss FSato KRondeel IScheltema RALuppens GfalseCAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands.During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitutions, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Polϵ) for PCNA binding. However, CAF-1 does not affect the activity of the lagging strand DNA poly2023-01-01T00:00:00Z2023 May2026-07-15T01:42:05.248Z2025-04-05T21:08:24.488ZS-EPMC101645773694248410.1093/nar/gkad171