Hst3 Is Regulated by Mec1-dependent Proteolysis and Controls the S Phase Checkpoint and Sister Chromatid Cohesion by Deacetylating Histone H3 at Lysine 56

Julian Simon,Benjamin Newcomb,Eric Foss,Jessica Kim,Antonio Bedalov,Safia Thaminy,Tonibelle Gatbonton

doi:10.1074/jbc.m706384200

Abstract

The SIR2 homologues HST3 and HST4 have been implicated in maintenance of genome integrity in the yeast Saccharomyces cerevisiae. We find that Hst3 has NAD-dependent histone deacetylase activity in vitro and that it functions during S phase to deacetylate the core domain of histone H3 at lysine 56 (H3K56). In response to genotoxic stress, Hst3 undergoes rapid Mec1-dependent phosphorylation and is targeted for ubiquitin-mediated proteolysis, thus providing a mechanism for the previously observed checkpoint-dependent accumulation of Ac-H3K56 at sites of DNA damage. Loss of Hst3-mediated regulation of H3K56 acetylation results in a defect in the S phase DNA damage checkpoint. The pathway that regulates H3K56 acetylation acts in parallel with the Rad9 pathway to transmit a DNA damage signal from Mec1 to Rad53. We also observe that loss of Hst3 function impairs sister chromatid cohesion (SCC). Both S phase checkpoint and SCC defects are phenocopied by H3K56 point mutants. Our findings demonstrate that Hst3-regulated H3K56 acetylation safeguards genome stability by controlling the S phase DNA damage response and promoting SCC.

Highlights

Unlike cells in genotoxic stress resistance and Ac-H3K56 deacetylation, released in medium lacking methyl methanesulfonate (MMS), which showed an increase in we found that point mutants in Hst3 known to abolish enzy- Hst3 level that peaked at 60 min, cells released in MMS did not
In order to determine whether hst3 hst4 mutants, like mutants in other intra-S phase DNA damage response genes (e.g. TOF1 and MRC1), exhibit endogenous DNA damage during DNA replication (12, 22), we evaluated the spontaneous formation of DNA damage-associated checkpoint protein foci in hst3 hst4 cells during S phase
In order to determine whether deacetylase activity of Hst3 and Hst4 is required for faithful sister chromatid cohesion (SCC), we evaluated SCC in wild type cells treated with 50 mM nicotinamide

Summary

Introduction

In order to determine whether the observed decline of Hst3 following S phase is mediated through proteasome degradation, cells were released into S phase from ␣ factor-induced G1 arrest into medium containing the proteasome inhibitor MG132, and Hst3 level was monitored by Western blot. Wild type and checkpoint-deficient mec1 cells were released into S phase from ␣ factor-induced G1 arrest in medium containing MMS and MG132.

Results

Conclusion