Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage - PubMed (original) (raw)

Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage

Christophe Redon et al. EMBO Rep. 2003 Jul.

Abstract

Cells maintain genomic stability by the coordination of DNA-damage repair and cell-cycle checkpoint control. In replicating cells, DNA damage usually activates intra-S-phase checkpoint controls, which are characterized by delayed S-phase progression and increased Rad53 phosphorylation. We show that in budding yeast, the intra-S-phase checkpoint controls, although functional, are not activated by the topoisomerase I inhibitor camptothecin (CPT). In a CPT-hypersensitive mutant strain that lacks the histone 2A (H2A) phosphatidylinositol-3-OH kinase (PI(3)K) motif at Ser 129 (h2a-s129a), the hypersensitivity was found to result from a failure to process full-length chromosomal DNA molecules during ongoing replication. H2A Ser 129 is not epistatic to the RAD24 and RAD9 checkpoint genes, suggesting a non-checkpoint role for the H2A PI(3)K site. These results suggest that H2A Ser 129 is an essential component for the efficient repair of DNA double-stranded breaks (DSBs) during replication in yeast, particularly of those DSBs that do not induce the intra-S-phase checkpoint.

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Figures

Figure 1

Figure 1

H2A(X) Ser 129 is essential for survival in the presence of topoisomerase-1-mediated DNA damage. (A) γ-H2A(X) formation by various DNA-damaging agents in wild-type (WT) yeast. Cultures were exposed to the agents for 1 h (14 min for IR) at 30 °C. (B) γ-H2A(X) formation in wild-type yeast incubated with various concentrations of camptothecin (CPT) or (C) incubated with 20 μM CPT for various durations. (D) γ-H2A(X) formation in strains in which Ser 129 had been altered to alanine in either one or both H2A(X) genes and which had been incubated with 20 μM CPT for 15 min. (E–G) h2a-s129a cells are sensitive to CPT. (E) Cells were treated with active (S) or inactive (R) CPT. Insertion of a functional HTA1 gene (F) and deletion of topoisomerase 1 (TOP1) (G) rescue the h2a-s129a mutation from CPT-induced death. Bleo, 20 μg bleomycin ml−1; C, untreated controls; H2A(X), histone 2A(X); IR, irradiation with 200 Gy; HU, 200 mM hydroxyurea; MMS, 0.2% methylmethanesulphonate; Ph, 2.5 μg phleomycin ml−1.

Figure 2

Figure 2

Cell-cycle analysis. Wild-type (WT) and h2a-s129a cells were synchronized in G1 phase with α-factor and then released in the presence of 20 μM camptothecin (CPT) or 0.03% methylmethanesulphonate (MMS), as indicated. Aliquots were taken at the indicated times for analysis by fluorescence microscopy (A), fluorescence-activated cell-sorting analysis (B–E) or immunoblotting with an anti-Rad53 polyclonal antibody (F). DMSO, dimethylsulphoxide; Rad53P, phosphorylated Rad53. 1C and 2C indicate haploid and diploid DNA content, respectively.

Figure 3

Figure 3

Pulsed-field gel electrophoresis analysis of yeast chromosomes. (A) G1-synchronized cultures were released in 20 μM camptothecin (CPT). Aliquots of equal volume were taken at the indicated times, mixed immediately with equal volumes of melted agarose, and formed into plugs. The plugs were subjected to pulsed-field gel electrophoresis. The ethidium-bromide-stained gel was imaged, and the image subjected to densitometry. (B,C) Time courses of density changes of the smallest and largest chromosome bands (chromosome (Chr) I and Chr IV/XII) from the pulsed-field gel. The insets show the gel image. The horizontal lines indicate the initial density (1X) and complete population doubling (2X). (D) Relative extent of chromosome doubling. The density at 80 min post-release relative to the initial density was calculated for each chromosomal DNA band in the four cultures and was plotted against chromosome size. The horizontal lines denote the initial density (1X) and complete population doubling (2X). The symbols used on the graph are the same as in (B) and (C). The slopes of the wild-type cultures are −0.023 without CPT and −0.035 with CPT. The slopes of the mutant cultures are −0.037 without CPT and −0.67 with CPT. h2A, h2a-s129a; Kbp, kilobase pairs; WT, wild type.

Figure 4

Figure 4

Interactions of h2a-s129a with other mutations. (A) γ-H2A(X) does not form in response to camptothecin (CPT) treatment in the mec1-1 tel1Δ strain. (B–D) Cultures of the indicated strains were synchronized in G1 and released in the presence of 20 μM CPT. At the times indicated, aliquots were removed, diluted and plated on YPD without CPT to determine viability. Strains are listed in the supplementary information online. (E) Cultures of the indicated strains were irradiated (IR) and examined for survival. H2A(X), histone 2A(X); WT, wild type.

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