Inactivation of Ku-mediated end joining suppresses mec1Delta lethality by depleting the ribonucleotide reductase inhibitor Sml1 through a pathway controlled by Tel1 kinase and the Mre11 complex

Abstract

RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Their lethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80 suppresses mec1Delta, but not rad53Delta, lethality. We show that suppression of mec1Delta lethality is not due to Ku--associated telomeric defects but rather results from the inability of Ku- cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1Delta lethality is also suppressed by lif1Delta, which like yku70Delta and yku80Delta, prevents nonhomologous end joining. The viability of yku70Delta mec1Delta and yku80Delta mec1Delta cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.

FIG. 1.

Deletion of YKU70 suppresses mec1Δ lethality. (A) mec1Δ lethality is suppressed in cells generated from diploids homozygous for yku70Δ. The diploid strains mec1Δ/MEC1 YKU70/YKU70, mec1Δ/MEC1 yku70Δ/YKU70, and mec1Δ/MEC1 yku70Δ/yku70Δ were sporulated and dissected. Tetrads were displayed vertically on a YPD plate and incubated at 30°C for 3 days. Four representative tetrads are shown for each dissection. The arrows show mec1Δ spore clones. (B) HU sensitivity of mec1Δ yku70Δ cells. Haploid wild-type (wt) cells were transformed with the 2μm URA3-marked plasmid expressing RNR1 (pRS426-RNR1). Subsequently, mec1Δ or yku70Δ and mec1Δ gene deletions were created. Tenfold serial dilutions of fresh stationary-phase cultures were plated on SD-Ura and on YPD plates containing 20 mM, 5 mM, or 2 mM HU and subsequently incubated for 3 days at 30°C. (C) Effects of mec1Δ on viability, silencing, and telomere length of the yku70Δ cells. (Left) Cells of the indicated genotypes were streaked multiple times on YPD plates. Cells from the first, second, and third restreaks are shown. (Middle) Telomeric position effect was assayed by 10-fold serial dilution of the culture cells on 5-fluoroorotic acid (5-FOA) plates. (Right) Telomere lengths of mec1Δ yku70Δ strains. (D) Mec1 is not required for the viability of yku70Δ cells. Haploid wild-type cells were transformed with the 2μm URA3-marked plasmid expressing MEC1 (pRS426-MEC1). Subsequently, yku70Δ, mec1Δ, or yku70Δ and mec1Δ gene deletions were created. Tenfold serial dilutions of fresh stationary-phase cultures were plated on SD-Ura and 5-FOA plates and incubated for 3 days at 30°C.

FIG. 2.

Tel1 and Mre11 are required for the mec1Δ lethality suppression by yku70Δ. (A) Telomere length analysis for rif2Δ in wild-type (wt), yku80Δ, and yku80Δ tel1Δ cells. Lane M, ladder DNA serving as size standard. (B) Mre11 is phosphorylated in yku70Δ and mec1Δ yku70Δ cells. The indicated strains contain a plasmid allowing the expression of either wild-type Mre11-ProtA (+) or the vector (−) (14). Protein extracts of the indicated strains were analyzed by Western blotting for protein phosphorylation. Arrows indicate the position of the basal and phosphorylated (*) Mre11-ProtA bands. MW, molecular mass.

FIG. 3.

Suppression of the end-joining function of yKu suppresses mec1Δ lethality. (A) Telomere size analysis of the telomeric defective/repair-proficient yku80-PF437,438AA mutant. Telomere size was examined in strains of the indicated genotypes carrying either a pRS413-YKU80 plasmid or a pRS413 plasmid expressing the mutant allele yku80-PF437,438AA (42). Lane M, ladder DNA serving as size standard. wt, wild type. (B) lif1Δ suppresses mec1Δ lethality. The mec1Δ/MEC1 lif1Δ/lif1Δ diploid strain was dissected. The presence of mec1Δ lif1Δ spores is indicated by arrows.

FIG. 4.

TEL1 mRNA level is slightly increased in yku80Δ cells. Total RNA was extracted from wild-type (wt), yku80Δ, and tel1Δ cells and processed for RT-PCR with the appropriate primers to measure TEL1 and ACT1 mRNA levels. The signals specific for the TEL1 transcripts were normalized to that of the actin signal. The TEL1 transcript level was arbitrarily set at 1 in the wild-type cells, and RNA levels in mutant cells were determined accordingly. Bar graphs represent an analysis of the results from three experiments.

FIG. 5.

Regulation of Sml1 protein amount in yku80Δ cells. Sml1 levels are reduced in yku80Δ cells. A chromosomally encoded YFP-SML1 was introduced in the indicated strains. Exponentially growing cells of the indicated strains were blocked in G₁ with alpha factor (left), in S phase with hydroxyurea (center), or in G₂ with nocodazole (right), and the Sml1 level was analyzed either by epifluorescence microscopy (A) or by Western blot analysis (B) with anti-GFP antibodies (after confirmation of the efficiency of the G₁, S, and G₂ arrests by microscopic analyses). The arrowheads indicate the positions of YFP-Sml1. In each case, a band cross-reacting with anti-GFP antibodies is used as a loading control and the amount of YFP-Sml1 was normalized to that of the wild-type YFP-Sml1 level. Each value corresponds to the average of the results from three independent experiments. (C) Haploid cells of the indicated genotypes were transformed with a 2μm plasmid expressing RNR1 (pRS426-RNR1), and the Sml1 protein amount was analyzed by Western blot analysis. Here again, in each case, a band cross-reacting with anti-GFP antibodies is used as a loading control and the amount of YFP-Sml1 was normalized to the wild-type YFP-Sml1 level. MW, size standard.

FIG. 6.

SML1 deletion suppresses mec1Δ lethality in different genetic contexts. SML1 deletion rescues mec1Δ rad9Δ yku70Δ (A), mec1Δ mre11Δ yku70Δ (B), and mec1Δ tel1Δ yku70Δ (C) lethality. (A, B, C) Tetrads from diploids homozygous for yku70Δ and heterozygous for mec1Δ (left) or mec1Δ sml1Δ (right) and either rad9Δ (A), mre11Δ (B), or tel1Δ (C) were dissected and analyzed for the presence of auxotrophic markers. Four tetrads are shown for each and are displayed vertically. Open squares indicate the yku70Δ single mutant. Diamonds indicate sml1Δ yku70Δ mutants. Circles indicate mec1Δ yku70Δ mutants. Hexagons indicate sml1Δ mec1Δ yku70Δ mutants. Squares indicate rad9Δ yku70Δ (A), mre11Δ yku70Δ (B), or tel1Δ yku70Δ (C) mutants. Crosses indicate rad9Δ sml1Δ yku70Δ (A), mre11Δ sml1Δ yku70Δ (B), or tel1Δ sml1Δ yku70Δ (C) mutants. Triangles indicate mec1Δ rad9Δ yku70Δ (A), mec1Δ mre11Δ yku70Δ (B), or mec1Δ tel1Δ yku70Δ (C) mutants. Hearts indicate mec1Δ rad9Δ sml1Δ yku70Δ (A), mec1Δ mre11Δ sml1Δ yku70Δ (B), or mec1Δ tel1Δ sml1Δ yku70Δ (C) mutants.

FIG. 7.

Mre11 foci are detected in yku80Δ cells. Mre11 foci were analyzed in asynchronously growing cells in the absence (−) or presence (+) of MMS (0.05%). Cells were analyzed by YFP fluorescence and phase microscopy. The numbers indicate the percentages of cells that contained Mre11 foci. At least 500 cells were analyzed for each strain. Upper panels, YFP fluorescence; lower panels, phase contrast. wt, wild type.