Csm3 and Tof1 are epistatic with H2A-S129 with respect to survival after treatment with DNA-damaging agents. (A) Growth of h2a, csm3Δ, and tof1Δ yeast strains was compared after exposure to HU (40 mm), UV (150 J/m²), IR (500 Gy), and CPT (20 μm). Serial dilutions (10-fold) were spotted onto YPAD plates and incubated for 2 days at 30°. The csm3Δ, tof1Δ, and h2a strains display the same degree of sensitivity to these DNA-damaging agents. An mec1Δ mutant serves as a positive control. (B) The csm3Δ tof1Δ, csm3Δ h2a double mutants, and the csm3Δ tof1Δ h2a triple mutant have the same sensitivity to CPT as the csm3Δ single mutant. h2a refers to hta1-S129A hta2-S129A double mutant.

Csm3 and Tof1 function downstream of H2A-S129. (A) Deletion of CSM3 or TOF1 does not prevent H2A phosphorylation. Immunoblot of H2A phosphoserine 129 levels in wt and mutant yeast strains after exposure to 20 μm CPT for 60 min at 30° (CPT) or 200 Gy (IR). (0 = mock treated cells). The coomassie-stained gels and the Western blots were imaged and the images subjected to densitometry. Numbers reported under each lane correspond to the ratio of signal at one position-phosphorylated H2A in the blot (γ) to the signal in the region corresponding to histone H4 in the stained gel (H4). No ratio was calculated for h2a-S129A mutant cells, as no phosphorylated H2A was detected in the blots. Quantitation of the H4 band in SDS–PAGE is easier than that of the H2A. As the core histone ratios are constant, using the H4 band to normalize sample loading is equivalent to using the H2A band. (B) Cell aliquots were also processed for fluorescence microscopy. Red represents DNA, detected with propidium iodide and white represents phosphorylated H2A, detected with antiphospho H2a-S129. h2a refers to hta1-S129A hta2-S129A double mutant.

The H2A-S129/TOF1/CSM3 epistasis group is independent of the RAD9 epistasis group. (A) Yeast strains lacking both RAD9 and one of several members of the H2A/CSM1/TOF1 epistasis group show the same degree of sensitivity to CPT. h2a refers to hta1-S129A hta2-S129A. (B) The drawing depicts a simplified model of the pathway tested. H2A refers to H2A-S129. Serial dilutions (10-fold) were spotted.

H2A-S129, MRC1, and RAD9 function in redundant pathway for the repair of double-strand breaks generated by topoisomerase I. (A–D) Serial dilutions (10-fold) of strains with the indicated genotypes were spotted onto YPAD plates without or with indicated amount of CPT. Plates were incubated at 30° for 2 days before pictures were taken. h2a refers to hta1-S129A hta2-S129A double mutant. (E) The drawing depicts a simplified model of the pathway tested. H2A refers to H2A-S129.

CSM3, TOF1, and H2A-S129 show synthetic-lethal interaction with MRC1 and RAD9. Four tetrads from each cross are shown. The circles in rad9Δ × mrc1Δ mark mrc1Δ rad9Δ spore segregants. The circles in rad9Δ h2a × csm3Δ tof1Δ h2a mark csm3Δ tof1Δ rad9Δ h2a spore segregants. The circles in rad9Δ h2a × tof1Δ h2a mark rad9Δ tof1Δ h2a spore segregants. The circles in mrc1Δ rad9Δ × tof1Δ mark mrc1Δ rad9Δ tof1Δ spore segregants. The circles in mrc1Δ rad9Δ × csm3Δ mark csm3Δ mrc1Δ rad9Δ spore segregants. The circles in rad9Δ h2a × mrc1Δ h2a mark mrc1Δ rad9Δ h2a spore segregants. h2a refers to hta1-S129A hta2-S129A double mutant. Tetrads were verified using independent markers and PCR. Colony genotypes are shown in Table 1. To simplify, only mutated genes are shown. c, csm3Δ; h, hta1-S129A hta2-S129A; m, mrc1Δ; t, tof1Δ; and 9, rad9Δ.

H2A-S129 is not required for efficient sister-chromatid cohesion. (A) Serial dilutions (10-fold) of strains with the indicated genotypes were spotted onto YPAD plates without (control) or with 12.5 μg/ml benomyl. Plates were incubated at 20° for 4 days before pictures were taken. (B) Sister-chromatid cohesion in h2a, csm3Δ, mrc1Δ mutants, and wt. Sister chromatid-cohesion assays were performed for chromosome III location in wild-type (wt), H2A mutant (h2a), csm3Δ, and mrc1Δ strains, counting 200 cells each time in three independent experiments and for each genotype. h2a refers to hta1-S129A hta2-S129A double mutant.

H2A-S129, DCC1, and RAD9 function in redundant pathways for the repair of DSBs generated by topoisomerase I. (A and B) Serial dilutions (10-fold) of strains with the indicated genotypes were spotted onto YPAD plates without or with indicated amount of CPT. Plates were incubated at 30° for 2 days before pictures were taken. h2a refers to hta1-S129A hta2-S129A double mutant. (C) The drawing depicts a simplified model of the pathway tested. H2A refers to H2A-S129.

Proposed genetic interactions involving CSM3, DCC1, H2A-S129, MRC1, RAD9, and TOF1. The drawing depicts a simplified model for the repair of CPT-induced replicative damage to illustrate the genetic pathway relationships explored in this study. H2A refers to H2A-S129.