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. 2013;9(1):e1003237.
doi: 10.1371/journal.pgen.1003237. Epub 2013 Jan 24.

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

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Free PMC article

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

Sandra Muñoz-Galván et al. PLoS Genet. .
Free PMC article

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.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Molecular analysis of SCR in 27 hyper-recombination mutants.
(A) Scheme of plasmid pRS316TINV used for the physical monitoring of SCR of a replication-born DSB. Fragments generated after XhoI-SpeI digestion, detected with a LEU2 probe (line with asterisks) are indicated with their corresponding sizes. SCR is monitored by appearance of the 4.7-kb fragment, the only one unequivocally occurring via SCR. (B) Quantification of DSB 2.4- and 1.4-kb fragments after 6 hours of HO activation in galactose. (C) Quantification of the SCR 4.7-kb fragment after 6 hours of HO activation in galactose. All strains, isogenic to W303, were transformed with pRS413GALHO harboring the HO endonuclease gene under the control of the GAL10 promoter, and pRS316TINV. Average and standard deviations of three samples from each genotype are plotted. Additional results are shown in Figures S1, S2, S3.
Figure 2
Figure 2. Molecular analysis of SCR in 13 SCR–defective mutants.
Kinetic analysis of SCR in 14 mutants (WS strains isogenic to W303) pre-selected as SCR-defective candidates. Representative genomic blots and quantification of HO-induced DSBs (upper panel) and SCE recombination (lower panel) are shown. Average and standard deviations of three samples from each genotype are plotted.
Figure 3
Figure 3. Genetic analysis of recombination in the 13 SCR–defective mutants.
(A) Analysis of Leu+ intrachromosomal recombination, as an indirect measure of unequal SCR, and plasmid-chromosome recombination events after 5 hr of HO activation in 2% galactose. Values plotted for each genotype are the average and standard deviations of the median of three independent fluctuation tests (each based on 6 samples) performed with three different transformants. (B) Analysis of spontaneous SCR in the chromosomal direct-repeat system his3-Δ5′::his3-Δ3′ in WT, hst3Δ, ahc1Δ, rtt109Δ, wss1Δ and rad52-C180A strains. A picture of the system and the expected His3+ recombination products are shown.
Figure 4
Figure 4. Effect of H3K56 acetylation/deacetylation on genetic SCR.
Genetic analysis of unequal SCR and plasmid-chromosome Leu+ recombination events after 5 hr of HO activation in isogenic wild-type (WS), hst3Δ, hst4Δ and hst3Δ hst4Δ strains.
Figure 5
Figure 5. Effect of H3K56 acetylation/deacetylation on molecular SCR.
(A) Physical analysis of SCR in isogenic wild-type (WS), hst3Δ, hst4Δ and hst3Δ hst4Δ strains after different times of HO induction (B) Physical analysis of SCR in isogenic wild-type, H3K56R and H3K56Q strains. Other details as in Figure 1.
Figure 6
Figure 6. H3K56 acetylation increases DNA damage.
Rad52 foci in different mutants affected in the histone H3K56 acetylation/deacetylation pattern. Average and standard deviation of two independent experiments are shown.
Figure 7
Figure 7. Effect of changes in the state of H3K56 acetylation on spontaneous recombination.
Analysis of spontaneous intrachromosomal and plasmid-chromosome recombination in isogenic wild-type (WS), hst3Δ, hst4Δ, hst4Δ hst3Δ and rad52-C180A strains.
Figure 8
Figure 8. Inviability/synthetic growth defect of histone H3K56 deacetylation mutants in the absence of Rad51 and Pol32.
Tetrad analysis of a rad51Δ hst4Δ hst3Δ x pol32Δ cross. Squares indicate quadruple mutants, which fail to grow.
Figure 9
Figure 9. Model to explain how the state of acetylation/deacetylation of H3K56 influences SCR.
Newly incorporated Histone H3 in the newly born sister-chromatids are acetylated, whereas the unreplicated DNA contains deacetylated histone H3. In the absence of K56 acetylation or when all histones H3 are acetylated, the recombination apparatus does not efficiently recognize the sister and the SCR preference is lost.

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