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

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 ubiquitinmediated 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.Post-translational modification of histone proteins has been implicated in controlling many aspects of chromosome biology. Among the recently described histone modifications is acetylation of histone H3 on lysine 56 (Ac-H3K56) located at the lateral surface of the nucleosome core (1-4). Ac-H3K56 appears in S phase on newly synthesized histone H3 prior to its deposition onto chromatin, and Lys 56 is deacetylated later in S phase following its deposition (5). Acetylation of H3K56 requires a histone chaperone, Asf1 (6, 7), and is carried out by a recently characterized acetyltransferase Rtt109 (8 -11). Regulation of acetylation is critical, since cells with a mutant form of histone H3 that cannot be acetylated are DNA damage-and hydroxyurea (HU) 3 -sensitive. In the presence of DNA damage, persistent checkpoint-dependent H3K56 acetylation suggested an interplay between classical DNA damage checkpoint proteins (e.g. Mec1) and regulators of H3K56 acetylation.In response to genotoxic stress, Mec1, acting as a DNA damage sensor, activates the DNA damage response network by phosphorylating Rad53. Rad9 serves as an adaptor (12) that facilitates Mec1-mediated phosphorylation of Rad53 throughout the cell cycle. In addition, and specifically during S phase, Mec1 is capable of efficiently phosphorylating Rad53 in a RAD9-independent fashion (13, 14). The ability of Mec1 to transmit a DNA damage signal during S phase in the absence of RAD9 depends on several genes (e.g. TOF1, CSM3, and MRC1) that constitute an S phase-specific branch of the DNA damage response network (15). In addition to DNA damage signal transduction from Mec1 to Rad53, the members of the S phasespecific branch of the DNA damage response network play a separate role in ensuring the stability of the stalled replication forks (16,17) and enabling the establishment of faithful sister chromatid cohesion SCC (18,19). Their role in SCC appears to be ...

Hst3 is turned over by a replication stress-responsive SCF Cdc4 phospho-degron

“…Previously, it was known that the increase in turnover seen in response to DNA damage required Mec1, the homolog of human ATR and an upstream activating kinase of both the DNA damage and replication checkpoint pathways (8). In response to both DNA damage and replication stress, the checkpoint kinase Mec1 phosphorylates and activates the downstream effector kinase Rad53.…”

“…During an unperturbed cell cycle, HST3 transcript levels cycle, and Hst3 protein turns over very quickly (5,9). In response to DNA damage, transcription of HST3 is turned down, and Hst3 protein turnover is even faster (5,8,(10)(11)(12).…”

“…During an unperturbed cell cycle, HST3 transcript levels cycle, and Hst3 protein turns over very quickly (5,9). In response to DNA damage, transcription of HST3 is turned down, and Hst3 protein turnover is even faster (5,8,(10)(11)(12).Many cell-cycle-regulated proteins are targeted for proteasomal degradation after ubiquitination by a member of the SCF E3 ubiquitin ligase family. SCF ligases are multisubunit ubiquitin ligases composed of Skp1, a cullin (Cdc53), a RING-finger protein (Rbx1), and an F-box protein (13).…”

“…In the budding yeast S. cerevisiae, Hst3 is a sirtuin histone deacetylase that, along with its close homolog Hst4, removes K56 acetylation (5,7,8). Overexpression of Hst3 or deletion of Hst3, along with related deacetylase Hst4, sensitizes cells to replication stress, suggesting that cells must control Hst3 levels precisely (5,7).…”

Nicotinamide Suppresses the DNA Damage Sensitivity of Saccharomyces cerevisiae Independently of Sirtuin Deacetylases

“…Hst3 and Hst4 deacetylate histone H3 on K56, which is deposited during DNA replication, and this deacetylation is prevented in the presence of DNA damage (Celic et al 2006;Maas et al 2006;Miller et al 2006;Edenberg et al 2014b). Levels of Hst3 and Hst4 are tightly controlled (Thaminy et al 2007;Delgoshaie et al 2014;Edenberg et al 2014b) and their absence results in persistent H3 K56 acetylation. Deregulation of H3 K56 acetylation is associated with spontaneous activation of the DNA damage response and defects in DNA repair (Celic et al 2006(Celic et al , 2008Maas et al 2006;Muñoz-Galván et al 2013;Che et al 2015;Simoneau et al 2015).…”