In budding yeast, acetylation of histone H3 lysine 56 (H3-K56) is catalyzed by the Rtt109-Vps75 histone acetyltransferase (HAT) complex, with Rtt109 being the catalytic subunit, and histone chaperone Asf1 is required for this modification. Cells lacking Rtt109 are susceptible to perturbations in DNA replication. However, how Asf1 regulates acetylation of H3-K56 and how loss of H3-K56 acetylation affects DNA replication are unclear. In vitro, here we show that at low concentrations, the Rtt109-Vps75 HAT complex acetylates H3-K56 when H3/H4 complexed with Asf1, but not H3/H4 tetramers, recapitulating the in vivo requirement of Asf1 for H3-K56 acetylation using recombinant proteins. Moreover, the Rtt109-Vps75 complex interacts with Asf1-H3/H4, but not Asf1. In vivo, Rtt109 interacts with Asf1 transiently and this interaction is abolished in the Asf1(V94R) mutant that loses its ability to bind H3/H4. Furthermore, the Rtt109 homolog in Schizosaccharomyces pombe (SpRtt109) also displayed an Asf1-dependent H3-K56 HAT activity in vitro. These results indicate that Asf1 regulates H3-K56 acetylation mainly through its effect on histones H3 and H4, and this effect is likely to be conserved. Lastly, we have shown that cells lacking Rtt109 or expressing H3-K56 mutants exhibited significant reduction in the association of three proteins with stalled DNA replication forks and hyperrecombination of replication forks stalled at replication fork barriers of the ribosomal DNA locus compared to wild type cells. Taken together, these studies provide novel insight into the role of Asf1 in the regulation of H3-K56 acetylation and the function of this modification in DNA replication.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|