Histone H2A‐H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin

Abstract

The eukaryotic replisome disassembles parental chromatin at DNA replication forks, but then plays a poorly understood role in the re‐deposition of the displaced histone complexes onto nascent DNA. Here, we show that yeast DNA polymerase α contains a histone‐binding motif that is conserved in human Pol α and is specific for histones H2A and H2B. Mutation of this motif in budding yeast cells does not affect DNA synthesis, but instead abrogates gene silencing at telomeres and mating‐type loci. Similar phenotypes are produced not only by mutations that displace Pol α from the replisome, but also by mutation of the previously identified histone‐binding motif in the CMG helicase subunit Mcm2, the human orthologue of which was shown to bind to histones H3 and H4. We show that chromatin‐derived histone complexes can be bound simultaneously by Mcm2, Pol α and the histone chaperone FACT that is also a replisome component. These findings indicate that replisome assembly unites multiple histone‐binding activities, which jointly process parental histones to help preserve silent chromatin during the process of chromosome duplication.

The replisome guides nucleosome assembly during DNA replication

“…It is worth mentioning that the mutation that abolished the histone-binding activity of Mcm2 disrupted the association between Pol α and Mcm2 without affecting the interaction of Pol α with Ctf4. Disrupting the H2A-H2B-binding site of Pol α led to similar results [148]. Early studies also showed that Pol α can bind FACT [151][152][153].…”

“…Moreover, disrupting the histone-binding ability of either Pol ε or Pol α impaired parental histone segregation on daughter strands, indicating a direct role of polymerase in RC nucleosome assembly. Supporting this idea, mutations disrupting the Pol α-H2A-H2B interaction and the Pol α-Ctf4 interaction led to silencing loss in telomeric regions and at mating type loci [148]. Pol α was also important for heterochromatin silencing maintenance in fission yeast and the restoration of repressive histone marks in plants [159,160].…”

“…In yeast, all core histones released from chromatin were detected in the Pol α-associated protein complex [148]. Moreover, H2A-H2B histone-binding sites have been identified in the N-terminal of the large subunit, Pol1, in both yeast and human cells [148]. In yeast, Pol α connects to CMG helicase via the homotrimer hub Ctf4 [149].…”

“…Intriguingly, the amount of FACT complex copurified with Pol α increased in the absence of Ctf4 [152]. Moreover, the interaction of Pol α with FACT was reduced by disrupting the H2A-H2Bbinding activity of Pol α [148]. It would be interesting to decipher the relationships among Mcm2, Ctf4, Polα, FACT and histones and to determine how these interactions are involved in parental histone H3-H4 recycling.…”

Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork

“…These include Ctf4 (And-1), Mrc1 (Claspin) and Csm3/Tof1 (Tipin/Timeless) that were identified as components of replisome progression complexes -stable CMG-containing complexes isolated from S-phase yeast cells (Gambus et al, 2006). Ctf4 is a trimeric hub that recruits proteins involved in sister chromatid cohesion, ribosomal DNA maintenance, parental histone transfer and gene silencing (Evrin et al, 2018;Gan et al, 2018;Samora et al, 2016;Simon et al, 2014;Villa et al, 2016). Csm3/Tof1 and Mrc1 are key replisome modulators, collectively termed the fork protection complex, that intimately associate with one another, both physically and functionally Katou et al, 2003).…”