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

Meas R, Smerdon MJ, Wyrick JJ - Nucleic Acids Res. (2015)

Bottom Line: 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.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.We observed increased levels of ubiquitylated PCNA and significantly lower mutation frequency in the tailless H2A/H3 mutant, indicating a defect in postreplication repair.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular Biosciences, Washington State University, Pullman, WA 99164-7520, USA.

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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 t0.5 (time to repair 50% of lesions) for its respective band. The smoothed lines were determined by averaging the t0.5 of 41 nucleotides surround each nucleotide site, where the 41 nucleotides encompass the nucleotide position and 20 nucleotides flanking that nucleotide in each direction.
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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 t0.5 (time to repair 50% of lesions) for its respective band. The smoothed lines were determined by averaging the t0.5 of 41 nucleotides surround each nucleotide site, where the 41 nucleotides encompass the nucleotide position and 20 nucleotides flanking that nucleotide in each direction.

Mentions: To determine if the hypersensitivity to MMS shown by the tH2A:tH3 mutant is due to a reduction in BER of alkylated bases in the mutant, we directly measured the overall repair of genomic DNA in the tailless histone strains after MMS treatment in the presence of nocodazole to arrest cells and prevent DNA replication. We found that the single tail deletions of H2A, H2B or H3 result in repair of MMS-induced lesions to a similar degree as that of WT cells in the genome overall (Figure 2A). Importantly, consistent with the MMS sensitivity results, the time course of MMS damage removal is significantly decreased in the tH2A:tH3 double tailless mutant, although repair is still evident (compare tH2A:tH3 mutant to mag1Δ in Figure 2A). In contrast, the tH2B:tH3 double tailless mutant had no detectable effect on MMS damage removal, indicating that the histone-associated repair defect is specifically linked with the combinatorial deletion of the tails of H2A and H3. By calculating the ensemble average DNA size on the alkaline gels, we determined the projected average fragment length for each repair time (Figure 2A, gels). As shown in the tH2A:tH3 gel (Figure 2A, middle gel panel), the ensemble average DNA size of the tH2A:tH3 mutant is significantly retarded compared to the WT. Moreover, this defective genomic BER in tH2A:tH3 mutant cells is not due to a difference in the amount of damage associated with the MMS exposure, as the initial levels of methylpurines/kb is similar amongst the various strains (Figure 2A, table).


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

Meas R, Smerdon MJ, Wyrick JJ - Nucleic Acids Res. (2015)

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 t0.5 (time to repair 50% of lesions) for its respective band. The smoothed lines were determined by averaging the t0.5 of 41 nucleotides surround each nucleotide site, where the 41 nucleotides encompass the nucleotide position and 20 nucleotides flanking that nucleotide in each direction.
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Related In: Results  -  Collection

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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 t0.5 (time to repair 50% of lesions) for its respective band. The smoothed lines were determined by averaging the t0.5 of 41 nucleotides surround each nucleotide site, where the 41 nucleotides encompass the nucleotide position and 20 nucleotides flanking that nucleotide in each direction.
Mentions: To determine if the hypersensitivity to MMS shown by the tH2A:tH3 mutant is due to a reduction in BER of alkylated bases in the mutant, we directly measured the overall repair of genomic DNA in the tailless histone strains after MMS treatment in the presence of nocodazole to arrest cells and prevent DNA replication. We found that the single tail deletions of H2A, H2B or H3 result in repair of MMS-induced lesions to a similar degree as that of WT cells in the genome overall (Figure 2A). Importantly, consistent with the MMS sensitivity results, the time course of MMS damage removal is significantly decreased in the tH2A:tH3 double tailless mutant, although repair is still evident (compare tH2A:tH3 mutant to mag1Δ in Figure 2A). In contrast, the tH2B:tH3 double tailless mutant had no detectable effect on MMS damage removal, indicating that the histone-associated repair defect is specifically linked with the combinatorial deletion of the tails of H2A and H3. By calculating the ensemble average DNA size on the alkaline gels, we determined the projected average fragment length for each repair time (Figure 2A, gels). As shown in the tH2A:tH3 gel (Figure 2A, middle gel panel), the ensemble average DNA size of the tH2A:tH3 mutant is significantly retarded compared to the WT. Moreover, this defective genomic BER in tH2A:tH3 mutant cells is not due to a difference in the amount of damage associated with the MMS exposure, as the initial levels of methylpurines/kb is similar amongst the various strains (Figure 2A, table).

Bottom Line: 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.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.We observed increased levels of ubiquitylated PCNA and significantly lower mutation frequency in the tailless H2A/H3 mutant, indicating a defect in postreplication repair.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular Biosciences, Washington State University, Pullman, WA 99164-7520, USA.

Show MeSH
Related in: MedlinePlus