Histone H3K56 Acetylation, Rad52, and Non-DNA Repair Factors Control Double-Strand Break Repair Choice with the Sister Chromatid

Abstract

DNA double-strand breaks (DSBs) are harmful lesions that arise mainly during replication. The choice of the sister chromatid as the preferential repair template is critical for genome integrity, but the mechanisms that guarantee this choice are unknown. Here we identify new genes with a specific role in assuring the sister chromatid as the preferred repair template. Physical analyses of sister chromatid recombination (SCR) in 28 selected mutants that increase Rad52 foci and inter-homolog recombination uncovered 8 new genes required for SCR. These include the SUMO/Ub-SUMO protease Wss1, the stress-response proteins Bud27 and Pdr10, the ADA histone acetyl-transferase complex proteins Ahc1 and Ada2, as well as the Hst3 and Hst4 histone deacetylase and the Rtt109 histone acetyl-transferase genes, whose target is histone H3 Lysine 56 (H3K56). Importantly, we use mutations in H3K56 residue to A, R, and Q to reveal that H3K56 acetylation/deacetylation is critical to promote SCR as the major repair mechanism for replication-born DSBs. The same phenotype is observed for a particular class of rad52 alleles, represented by rad52-C180A, with a DSB repair defect but a spontaneous hyper-recombination phenotype. We propose that specific Rad52 residues, as well as the histone H3 acetylation/deacetylation state of chromatin and other specific factors, play an important role in identifying the sister as the choice template for the repair of replication-born DSBs. Our work demonstrates the existence of specific functions to guarantee SCR as the main repair event for replication-born DSBs that can occur by two pathways, one Rad51-dependent and the other Pol32-dependent. A dysfunction can lead to genome instability as manifested by high levels of homolog recombination and DSB accumulation.

Regulation of ribosomal DNA amplification by the TOR pathway

“…S5A). H3 K56R mutants displayed a stronger phenotype than H3 K56Q mutants; however, this difference is not specific to rDNA recombination; promotion of HRdependent sister chromatid recombination depends on an H3 K56 acetylation-deacetylation cycle and is more seriously impaired by H3 K56R than H3 K56Q mutations (34). Recent genetic evidence also shows that although hypo-and hyperacetylation of H3 K56 both inhibit HR, they do so through different mechanisms (35).…”

“…Sir2 regulates expression of ncRNAs in the rDNA spacer, removing cohesin and allowing a broken replication fork to undergo BIR with unmatched repeats (10). In contrast, Hst3 and Hst4 control recombination pathway choice at stalled replication forks; disturbance of the H3 K56 acetylation cycle prevents HR with a sister chromatid (34), and at the rDNA instigates non-HR-dependent recombination, leading to amplification. Because the non-HR-dependent pathway causes constitutive gain of rDNA copies, cells could regulate rDNA amplification by modulating H3 K56 acetylation (see model Fig.…”

Histone modifications in DNA damage response

“…During the S phase of cell cycle, H3K56Ac is on the newly synthesized histone H3 that is incorporated into chromosomes (Masumoto et al, 2005), and it drives chromatin reassembly and checkpoint recovery after DNA repair with the assistance of histone chaperone Asf1 Driscoll et al, 2007). H3K56Ac abrogation results in sensitivity to genotoxic agents that cause DNA strand breaks, genome instability and decreased sister chromatid recombination in the S phase (Munoz-Galvan et al, 2013;Wurtele et al, 2012). However, cells proceed into G2 phase after DNA replication fork damage repair is completed, and H3K56 acetylation largely disappears in G2 phase (Masumoto et al, 2005).…”

Multi-BRCT scaffolds use distinct strategies to support genome maintenance

“…97 Indeed, removing Rtt109 or the Rtt101 complex impairs recombinational repair and replication, and sensitizes cells to replication stress. 93,94,[98][99][100][101] Once replication is completed, histone deacetylases Hst3 and Hst4 remove the acetyl group from H3K56ac to establish a more stable nucleosome state. [102][103][104] Cells lacking Hst3 and Hst4 or containing the acetylation mimetic H3 mutation (H3K56Q) exhibit defects such as persistent checkpoint and sensitivity to higher temperature and genotoxins.…”