The RAD6/BRE1 histone modification pathway in Saccharomyces confers radiation resistance through a RAD51-dependent process that is independent of RAD18

Abstract

We examine ionizing radiation (IR) sensitivity and epistasis relationships of several Saccharomyces mutants affecting post-translational modifications of histones H2B and H3. Mutants bre1Delta, lge1Delta, and rtf1Delta, defective in histone H2B lysine 123 ubiquitination, show IR sensitivity equivalent to that of the dot1Delta mutant that we reported on earlier, consistent with published findings that Dot1p requires H2B K123 ubiquitination to fully methylate histone H3 K79. This implicates progressive K79 methylation rather than mono-methylation in IR resistance. The set2Delta mutant, defective in H3 K36 methylation, shows mild IR sensitivity whereas mutants that abolish H3 K4 methylation resemble wild type. The dot1Delta, bre1Delta, and lge1Delta mutants show epistasis for IR sensitivity. The paf1Delta mutant, also reportedly defective in H2B K123 ubiquitination, confers no sensitivity. The rad6Delta, rad51null, rad50Delta, and rad9Delta mutations are epistatic to bre1Delta and dot1Delta, but rad18Delta and rad5Delta show additivity with bre1Delta, dot1Delta, and each other. The bre1Delta rad18Delta double mutant resembles rad6Delta in sensitivity; thus the role of Rad6p in ubiquitinating H2B accounts for its extra sensitivity compared to rad18Delta. We conclude that IR resistance conferred by BRE1 and DOT1 is mediated through homologous recombinational repair, not postreplication repair, and confirm findings of a G1 checkpoint role for the RAD6/BRE1/DOT1 pathway.

Figure 1.—

Survival vs. X-ray dose for haploid mutant strains separately affected in methylation of three sites on histone H3. Two set2Δ strains, blocked in H3 K36 methylation, are compared to swd1Δ and dot1Δ strains, blocked in H3 K4 and H3 K79 methylation, respectively. A wild-type and a rad51Δ haploid strain are included for comparison. The strains share the same genetic background, and the dot1Δ mutant shows X-ray sensitivity equivalent to that of previously published dot1Δ strains in this background (Game et al. 2005).

Figure 2.—

Survival vs. X-ray dose for two bre1Δ and two lge1Δ haploid deletion strains. Wild-type, rad51Δ, and dot1Δ haploids are included for comparison. In addition, a curve for the bre1Δ strain g1329-26A transformed with a plasmid containing the BRE1 gene and its native promoter (pJB200 from James Brown at Stanford University) is shown.

Figure 3.—

Survival vs. X-ray dose for two rtf1Δ and two paf1Δ haploid deletion strains. Wild-type, rad51Δ, and dot1Δ haploids are included for comparison.

Figure 4.—

Survival vs. X-ray dose for two bre1Δ lge1Δ double-mutant haploid deletion strains, shown with representative bre1Δ and lge1Δ single mutants (see Figure 2). Wild-type and rad51Δ haploids are included for comparison.

Figure 5.—

Survival vs. X-ray dose for two bre1Δ dot1Δ double-mutant haploid deletion strains, shown with representative bre1Δ and dot1Δ single mutants (see Figure 2). Wild-type and rad51Δ haploids are included for comparison.

Figure 6.—

Survival vs. X-ray dose for three lge1Δ dot1Δ double-mutant haploid deletion strains and two bre1Δ lge1Δ dot1Δ triple-mutant strains, shown with a representative bre1Δ and lge1Δ single mutant (see Figure 2). Wild-type and rad51Δ haploids are included for comparison.

Figure 7.—

Survival vs. X-ray dose for dot1Δ rad6Δ, dot1Δ rad51∷URA3 and two rad6Δ rad51∷URA3 double-mutant haploid deletion strains together with dot1Δ, rad6Δ, and rad51∷URA3 single mutants and a wild-type strain. A rad51Δ BY4742 strain carrying the standard deletion library replacement cassette, which exhibits IR sensitivity equivalent to that conferred by the rad51∷URA3 disruption allele, is also shown.

Figure 8.—

Survival vs. X-ray dose for a bre1Δ rad18Δ double-mutant strain compared to two bre1Δ and two rad18Δ single mutants. A wild-type and a rad6Δ strain are included for comparison. This is the median rad6Δ curve of seven obtained; see text.

Figure 9.—

Survival vs. X-ray dose for two dot1Δ rad18Δ and one lge1Δ rad18Δ double-mutant strain compared to dot1Δ, lge1Δ, and rad18Δ single mutants. A wild-type and a rad6Δ strain are included for comparison.

Figure 10.—

Survival vs. X-ray dose for two dot1Δ lge1Δ rad18Δ and one dot1Δ bre1Δ rad18Δ triple-mutant strain and a dot1Δ bre1Δ lge1Δ rad18Δ quadruple mutant. These strains are compared with wild type, the four component single mutants, and two rad6Δ strains representing the most and least sensitive full curves of seven rad6Δ curves obtained; see text.

Figure 11.—

Survival vs. X-ray dose for two ubr1Δ haploid deletion strains and two bre1Δ ubr1Δ double mutants, compared with two ubr1Δ and two bre1Δ single mutants. A wild-type and a rad51Δ haploid strain are also shown.

Figure 12.—

Survival vs. X-ray dose for two haploid ubr1Δ rad18Δ double mutants and two bre1Δ ubr1Δ rad18Δ triple-mutant strains, compared with two rad18Δ and two ubr1Δ single mutants. A wild-type strain, a bre1Δ rad18Δ double mutant, and a rad6Δ strain (see also Figure 8) are also shown.

Figure 13.—

Survival vs. X-ray dose for two haploid rad5Δ mutant strains, a bre1Δ rad5Δ double mutant and a dot1Δ rad5Δ double mutant. A bre1Δ and a dot1Δ mutant are shown for comparison, together with wild type and a rad51Δ strain.

Figure 14.—

Survival vs. X-ray dose for a rad5Δ rad18Δ double mutant and a dot1Δ rad5Δ rad18Δ triple mutant, compared to the component single mutants and a dot1Δ rad18Δ and dot1Δ rad5Δ double mutant (see also Figures 9 and 13). A wild type and a rad51Δ curve are also shown.

Figure 15.—

Survival vs. X-ray dose for two rad50Δ mutants and a dot1Δ rad50Δ and a bre1Δ rad50Δ double mutant. A rad51Δ mutant, a wild type, and the dot1Δ and bre1Δ single mutants are included for comparison.

Figure 16.—

Survival vs. X-ray dose for two dot1Δ rad9Δ double-mutant strains and a dot1Δ bre1Δ rad9Δ triple mutant, compared to two rad9Δ single mutants. Wild type and the bre1Δ and dot1Δ single mutants are included for comparison.

Figure 17.—

Survival vs. X-ray dose for two rad9Δ rad51∷URA3 double mutants and one rad9Δ rad6Δ double-mutant and two rad9Δ rad6Δ rad51∷URA3 triple-mutant strains, compared with a rad6Δ rad51∷URA3 double mutant (see also Figure 5), a wild type, and the three component single mutants.

Figure 18.—

Survival vs. X-ray dose for two rad9Δ rad5Δ and one rad9Δ rad18Δ double mutant and two rad9Δ rad5Δ rad18Δ triple mutants. Each single mutant and wild type are also shown for comparison.

Figure 19.—

Effect on the IR-induced G₂/M checkpoint of the bre1Δ, dot1Δ, and rad9Δ mutations compared to wild type. Cells were released from nocodazole synchronization into fresh medium at time zero after 500 Gy of ¹³⁷Cs gamma irradiation, and in parallel without irradiation. The percentage of cells that have undergone nuclear division is shown for each culture at 30-min intervals thereafter. Strains used were g1295-21C bre1Δ, g1295-6C dot1Δ, g1304-19C rad9Δ, and BY4741 wild-type MATa.

Figure 20.—

Effect on the IR-induced G₁/S checkpoint of the bre1Δ, dot1Δ, and rad9Δ mutations compared to wild type. Cells were released from α-factor synchronization into fresh medium at time zero after 500 Gy of ¹³⁷Cs gamma irradiation, and in parallel without irradiation, and fixed and analyzed for DNA content with flow cytometry at 15-min intervals thereafter. Strains used for each genotype were as in Figure 19.