DNA Polymerase Delta Governs Parental Histone Transfer to DNA Replication Lagging Strand

Chromatin replication is intricately intertwined with DNA replication, and the recycling of parental histones is essential for epigenetic inheritance. The transfer of parental histones to both the DNA replication leading and lagging strands involves two distinct pathways: the leading strand utilizes DNA polymerase e subunits Dpb3/Dpb4, while the lagging strand is facilitated by the MCM helicase subunit Mcm2. However, the process by which Mcm2, moving along the leading strand, facilitates the transfer of parental histones to the lagging strand remains unclear. Our study reveals that the deletion of Pol32, a non-essential subunit of major lagging-strand DNA polymerase d, results in a predominant transfer of parental histone H3-H4 to the replication leading strand. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted by the mutation of Mcm2's histone H3-H4 binding domain. In conclusion, our findings identify the DNA polymerase delta subunit Pol32 as a novel key histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication. Overall design: We performed eSPAN according to the protocol in our previous studies with some modifications. Briefly, yeast cells were synchronized with alpha-factor and released in YPD medium with BrdU (400ug/ml) following the standard protocol. The cells were digested with MNase (NEB, Cat# M0247S), after saving 1% for BrdU-IP, the rest of digested chromatin was subjected to ChIP against H3K4me3 (ab8580), H3K56ac, HA-Tag (BETHYL,Cat#12CA5), or T7-Tag (BETHYL,Cat#A190-117A), individually. Then DNA extracted from the ChIP products was end-repaired and ligated with adapters using the VAHTS® Universal DNA Library Prep Kit for Illumina V3 (Vazyme, Cat#ND607). After saving 1% for ChIP-seq analysis, the products was subjected to BrdU-IP as previous reported, and the products was amplified with NEBNext High-Fidelity 2X PCR Master Mix (NEB, Cat# M0541L) and indexing primers. The obtained libraries were sequenced using the paired-end method by Illumina nova 6000.