NuA4 acetyltransferase is required for efficient nucleotide excision repair in yeast

Abstract

The nucleotide excision repair (NER) pathway is critical for removing damage induced by ultraviolet (UV) light and other helix-distorting lesions from cellular DNA. While efficient NER is critical to avoid cell death and mutagenesis, NER activity is inhibited in chromatin due to the association of lesion-containing DNA with histone proteins. Histone acetylation has emerged as an important mechanism for facilitating NER in chromatin, particularly acetylation catalyzed by the Spt-Ada-Gcn5 acetyltransferase (SAGA); however, it is not known if other histone acetyltransferases (HATs) promote NER activity in chromatin. Here, we report that the essential Nucleosome Acetyltransferase of histone H4 (NuA4) complex is required for efficient NER in Saccharomyces cerevisiae. Deletion of the non-essential Yng2 subunit of the NuA4 complex causes a general defect in repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast; in contrast, deletion of the Sas3 catalytic subunit of the NuA3 complex does not affect repair. Rapid depletion of the essential NuA4 catalytic subunit Esa1 using the anchor-away method also causes a defect in NER, particularly at the heterochromatic HML locus. We show that disrupting the Sds3 subunit of the Rpd3L histone deacetylase (HDAC) complex rescued the repair defect associated with loss of Esa1 activity, suggesting that NuA4-catalyzed acetylation is important for efficient NER in heterochromatin.

Keywords: Acetylation; Chromatin; DNA repair; Esa1; Histone; NER.

Figure 1.

Effects of deletion mutants in non-essential subunits of SAGA (gcn5Δ), NuA4 (yng2Δ), and NuA3 (sas3Δ) on repair of UV-induced CPD lesions in yeast. (A) Representative alkaline gels of genomic DNA isolated at the indicated repair time points following UV irradiation (100 J/m²), following treatment with or without (+/−) T4 endonuclease V. T4 endonuclease V creates single-stranded DNA nicks at CPD lesions, which were resolved on denaturing alkaline gels. (B-D) Quantification of CPD repair in mutant and wild-type (WT) yeast strains. The percentage of CPD lesions removed at each time point is plotted as the mean ± SEM of at least 3 independent experiments. P values were calculated using an unpaired t-test. *P ≤ 0.05.

Figure 2.

Rapid nuclear depletion of FRB-tagged Esa1 (AA-Esa1) using the anchor-away system. (A) Schematic diagram of the anchor-away system for rapidly depleting proteins from the nucleus. (B) Histone H4 acetylation is significantly diminished following anchor-away depletion of Esa1, based on western blot analysis of AA-Esa1 or isogenic wild type cells before and after 3 hours rapamycin treatment using a panH4acetyl antibody. Where indicated, a plasmid containing wild type Esa1 (+pEsa1) or mutant Esa1 (+pEsa1 E338Q) was co-expressed. GAPDH was used as a loading control. (C) Ten-fold serial dilutions of yeast spot onto synthetic complete (SC) or SC+ 1μg/mL rapamycin plates. (D) OD₆₀₀ measurements at the indicated time points following treatment with rapamycin. (E) Cultures treated with rapamycin for the indicated times were plated onto complete media and resultant colonies were counted and normalized to the untreated control. (D-E) represent the mean and SEM of 3 independent experiments.

Figure 3.

Anchor-away depletion of Esa1 causes a significant defect in NER of UV-induced CPD lesions. A) Representative alkaline gels of genomic DNA isolated at the indicated repair time points following UV irradiation (100 J/m²), following treatment with or without (+/−) T4 endonuclease V. Cells were preincubated with 1μg/ml rapamycin for 3 hours prior to UV irradiation to deplete Esa1 from the nucleus (AA-Esa1 strain), and were further incubated with rapamycin during the repair time course. A control strain (WT), in which Esa1 was not anchor-away tagged, was treated similar. (B-C) Quantification of %CPD repair from alkaline gels analyzing repair in the control (WT) or Esa1 anchor-away tagged (Esa1-AA) strain, treated with (B) or without (C) rapamycin. The mean ± SEM is depicted for a minimum of 3 independent replicates. P values were calculated for using an unpaired t-test. *P ≤ 0.05.

Figure 4.

Esa1 anchor-away depletion causes a significant repair defect at the heterochromatic HML locus, but not the actively transcribed RPB2 gene. Southern blot analysis for site specific NER of UV-induced CPD lesions at HML (A, B) and RPB2 (C, D) when Esa1 is conditionally depleted from the nucleus in the anchor-away system (see Figure 3 above). Quantification of % CPD repair at each locus is depicted as the mean and SEM for at least three independent replicates. P values were calculated using an unpaired t-test. Mean ± SEM is depicted. *P ≤ 0.05.

Figure 5.

Deletion of the Eaf1 component of the NuA4 complex causes a marginal defect in repair of CPD lesions. (A) Schematic representation of the NuA4 complex with the Piccolo NuA4 subcomplex shown in orange and the scaffolding component of the NuA4 complex shown in green. The subunits analyzed in this study (Yng2, Esa1, Eaf1) are highlighted with darker coloring. Diagram adapted from [45]. (B-C) NER phenotype of the eaf1Δ mutant. (B) A representative alkaline gel of CPD repair following UV irradiation. (C) Quantification of % repair for a minimum of 3 independent replicates, depicted as mean ± SEM.

Figure 6.

Disruption of the Rpd3L HDAC complex (i.e., sds3Δ), but not the Hos2 HDAC, suppresses the repair defect in NuA4 mutant cells. (A) Representative alkaline gels analyzing repair of CPD lesions in WT, esa1Δsds3Δ, and yng2Δhos2Δ mutants. (B-C) Quantification of NER alkaline gel analysis of HAT/HDAC double mutants esa1Δsds3Δ and yng2Δhos2Δ. Quantification represents the mean ± SEM of at least three independent replicates. P values were calculated using an unpaired t-test. *P ≤ 0.05.