The amino-terminal tails of histones H2A and H3 coordinate efficient base excision repair, DNA damage signaling and postreplication repair in Saccharomyces cerevisiae.
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.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.
Affiliation: School of Molecular Biosciences, Washington State University, Pullman, WA 99164-7520, USA.Show MeSH
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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).
Affiliation: School of Molecular Biosciences, Washington State University, Pullman, WA 99164-7520, USA.