The amino-terminal tails of histones H2A and H3 coordinate efficient base excision repair, DNA damage signaling and postreplication repair in Saccharomyces cerevisiae

Abstract

Histone amino-terminal tails (N-tails) are required for cellular resistance to DNA damaging agents; therefore, we examined the role of histone N-tails in regulating DNA damage response pathways in Saccharomyces cerevisiae. Combinatorial deletions reveal that the H2A and H3 N-tails are important for the removal of MMS-induced DNA lesions due to their role in regulating the basal and MMS-induced expression of DNA glycosylase Mag1. Furthermore, overexpression of Mag1 in a mutant lacking the H2A and H3 N-tails rescues base excision repair (BER) activity but not MMS sensitivity. We further show that the H3 N-tail functions in the Rad9/Rad53 DNA damage signaling pathway, but this function does not appear to be the primary cause of MMS sensitivity of the double tailless mutants. Instead, epistasis analyses demonstrate that the tailless H2A/H3 phenotypes are in the RAD18 epistasis group, which regulates postreplication repair. We observed increased levels of ubiquitylated PCNA and significantly lower mutation frequency in the tailless H2A/H3 mutant, indicating a defect in postreplication repair. In summary, our data identify novel roles of the histone H2A and H3 N-tails in (i) regulating the expression of a critical BER enzyme (Mag1), (ii) supporting efficient DNA damage signaling and (iii) facilitating postreplication repair.

Figure 1.

The N-tails of the canonical histones contribute to MMS sensitivity. (A) WT and histone N-tail deletion mutants were serially spotted onto YPD plates containing varying concentrations of MMS. A mag1Δ mutant is used to show MMS-hypersensitivity. (B) Cell survival assay of the tailless histone mutants from (A).

Figure 2.

The tH2A:tH3 mutant has reduced repair of methylpurines. In these experiments, the cells were arrested with nocodazole to prevent entry into S-phase, treated with 0.2% MMS for 10 min and then released into YPD containing nocodazole. (A) Genomic BER assays were performed on the tailless histone mutants. Representative alkaline gels for WT, tH2A:tH3 and mag1Δ cells are shown for DNA samples treated in the absence or presence (+/−) of AAG and APE1, which create single stranded nicks at 3-methyladenine and 7-methylguanine. The average fragment length is denoted for each time point (, WT; , tH2A:tH3; and , mag1Δ). The % of methylpurines removed at each time (or % repair) is shown in the graph as mean ± standard deviation of at least three independent experiments. The initial methylpurines/kb are listed in the table. (B) Repair was analyzed at the GAL10 locus by Southern blotting using locus-specific probes. Graph depicts mean ± standard deviation of at least three independent experiments. (C) High resolution DNA damage mapping was performed on the NTS of RPB2 for WT and tH2A:tH3 cells. The top band on the sequencing gel represents the full-length band, while bands that migrate faster represent methylpurine damage sites that have been cleaved by AAG and APE1 digestion. The arrow on the left indicates the transcription start site and the ovals on the right indicate the most prominent nucleosome positions along RPB2 as mapped by Jiang et al. (34). The plot is aligned so that each point is indicative of the t_0.5 (time to repair 50% of lesions) for its respective band. The smoothed lines were determined by averaging the t_0.5 of 41 nucleotides surround each nucleotide site, where the 41 nucleotides encompass the nucleotide position and 20 nucleotides flanking that nucleotide in each direction.

Figure 3.

Mag1 protein expression and MAG1 mRNA expression are reduced in tH2A:tH3 cells. WT and the N-tail deletion mutants were treated with 0.2% MMS for 10 min and further analyzed at the indicated time points. (A) Mag1^9myc and GAPDH were detected via western blotting. (B) A northern blot detecting MAG1 and reprobed for RDN18. [We note that the small band shift between the tH3(60’) sample and the tH2A:tH3 samples is due to a gel running artifact]. (C) Quantification of different gels, such as shown in (B) by normalizing MAG1 levels to that of WT in the absence of MMS. Error bars indicate standard deviations derived from three independent experiments.

Figure 4.

Overexpression of MAG1 enhances repair but does not suppress the MMS sensitivity in the tH2A:tH3 mutant. (A) Representative alkaline gels analyzing BER in genomic DNA were performed similarly to those in Figure 2. (B) The quantification of different gels, such as shown in (A), and the initial methylpurines/kb are shown in the table. (C) The vector only control (pVector) or the constitutively expressed MAG1 vector (pMAG1) was transformed into mag1Δ, WT and tH2A:tH3 cells and serially spotted onto sc-ura plates containing different MMS concentrations.

Figure 5.

The N-tail of H3 is important for proper Rad53 phosphorylation after DNA damage. Western blot detecting Rad53 and its hyperphosphorylated form (Rad53-P) before and after treatment with 0.2% MMS for 10 min.

Figure 6.

The N-tails of H2A and H3 are epistatic to RAD18. (A) Epistasis studies of (i) N-tail deletion mutants combinatorially deleted for (ii) MAG1, (iii) REV3, (iv) RAD5 and (v) RAD18. (B) Epistasis study of N-tail deletion mutants combinatorially deleted for RAD52. (C) MMS survival assay of WT and N-tail mutants (solid lines) and the N-tail mutants combinatorially deleted for RAD18 (dashed lines). (D) Growth curves of the cells in (C) in the absence of MMS. (E) Western blot of PCNA^9myc in WT and N-tail mutants before UV exposure and after a 1 h recovery in YPD following 100 J/m² of UV irradiation. GAPDH is used as the loading control. rad6Δ serves as a negative control for PCNA ubiquitylation. (F) Quantifications of (E) by normalizing (PCNA^9myc-ub)/(PCNA^9myc-total) and (PCNA^9myc-ub₂)/(PCNA^9myc-total) to WT. Error bars indicate standard deviations from three independent experiments.

Figure 7.

A model highlighting the contributions of H2A and H3 N-tails through different branches of MMS DNA tolerance. The N-tails of all histones within the nucleosome core particle (pdb: 1KX5; MacPyMOL) are represented in the ‘spheres’ configuration. The H3 N-tail is important for the regulation of DNA damage cell cycle checkpoint signaling via Rad9, which facilitates the subsequent hyperphosphorylation of Rad53. H2A and H3 N-tails are also important for upregulating BER gene expression by influencing unknown effectors (?) downstream of Rad53 to regulate Mag1. Furthermore, our epistasis and mutagenesis studies indicate the inclusion of the N-tails of H2A and H3 in the Rad18 epistasis group by influencing postreplication repair through TLS and possibly damage avoidance (?).