Summary Although essential for epigenetic inheritance, the transfer of parental histone (H3-H4)2 tetramers that contain epigenetic modifications to replicating DNA strands is poorly understood. Here, we show that the Mcm2-Ctf4-Polα axis facilitates the transfer of parental (H3-H4)2 tetramers to lagging-strand DNA at replication forks. Mutating the conserved histone-binding domain of the Mcm2 subunit of the CMG (Cdc45-MCM-GINS) DNA helicase, which translocates along the leading-strand template, results in a marked enrichment of parental (H3-H4)2 on leading-strand, due to the impairment of the transfer of parental (H3-H4)2 to lagging strands. Similar effects are observed in Ctf4 and Polα primase mutants that disrupt the connection of the CMG helicase to Polα that resides on lagging strand template. Our results support a model whereby parental (H3-H4)2 complexes displaced from nucleosomes by DNA unwinding at replication forks are transferred by the CMG-Ctf4-Polα complex to lagging-strand DNA for nucleosome assembly at the original location.

How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication–linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.