Mrc1 is required for sister chromatid cohesion to aid in recombination repair of spontaneous damage

Abstract

The SRS2 gene of Saccharomyces cerevisiae encoding a 3'-->5' DNA helicase is part of the postreplication repair pathway and functions to ensure proper repair of DNA damage arising during DNA replication through pathways that do not involve homologous recombination. Through a synthetic gene array analysis, genes that are essential when Srs2 is absent have been identified. Among these are MRC1, TOF1, and CSM3, which mediate the intra-S checkpoint response. srs2 Delta mrc1 Delta synthetic lethality is due to inappropriate recombination, as the lethality can be suppressed by genetic elimination of homologous recombination. srs2 Delta mrc1 Delta synthetic lethality is dependent on the role of Mrc1 in DNA replication but independent of the role of Mrc1 in a DNA damage checkpoint response. mrc1 Delta, tof1 Delta and csm3 Delta mutants have sister chromatid cohesion defects, implicating sister chromatid cohesion established at the replication fork as an important factor in promoting repair of stalled replication forks through gap repair.

FIG. 1.

SRS2 genetic interactions. Genome-wide synthetic genetic screens with srs2Δ identify genes involved in DNA repair and sister chromatid cohesion. The results of synthetic genetic array analysis with srs2Δ are presented as a genetic interaction map. Lines connecting genes represent synthetic lethality or synthetic slow growth. Colored circles designate the cellular role of the interacting genes.

FIG. 2.

srs2Δ synthetic interaction with mrc1Δ, tof1Δ, and csm3Δ. (A) The srs2Δ synthetic interactions are suppressed by loss of homologous recombination with a rad51Δ mutation. Five tetrads from each cross are shown. The circles mark the double mutants srs2Δ mrc1Δ, srs2Δ tof1Δ, and srs2Δ csm3Δ. The squares mark the triple mutants srs2Δ mrc1Δ rad51Δ, srs2Δ tof1Δ rad51Δ, and srs2Δ csm3Δ rad51Δ. In each cross, the triple mutant grew better than the double mutant. The strains used were HKY1307-12C (srs2Δ rad51Δ), HKY1253-15C (mrc1Δ), HKY1375-1D (tof1Δ), and HKY1374-2A (csm3Δ). (B) The circles mark the double mutant srs2K41A mrc1Δ. The srs2K41A mutant is mutated in the Walker A box, is defective in ATP binding, and has no DNA helicase activity or Rad51 filament removal activity (52). The strains used were HKY1435 (srs2K41A) and HKY1336-9B (mrc1Δ).

FIG. 3.

High-copy-number expression of Rad51 reduces the viability of an mrc1Δ strain. Transformants carrying a GAL1-RAD51 plasmid were streaked on SC-glucose or SC-galactose plates lacking leucine. The control galactose plate shows transformants with an empty GAL1 vector to verify that the mrc1Δ mutant was defective in growth on galactose-containing medium. The strains used were HKY1093-5A (MRC1) and HKY1235-15C (mrc1Δ).

FIG. 4.

Intrachromasomal recombination assay. Each strain carried the recombination reporter leu2-ri::URA3::leu2-bsteii, which has a heteroallelic duplication of LEU2, with URA3 between the LEU2 genes. Gene conversion was determined by fluctuation tests, measuring Leu⁺ Ura⁺ rates. The deletion rate was determined by fluctuation tests, measuring fluoroorotic acid resistance rates. Each test was performed with nine colonies and done three times for each genotype, with three spore segregants for each genotype. The strains used were HKY1538-2C, HKY1538-6A, and HKY1538-10A for the wild type, HKY1505-1C, HKY1505-6B, and HKY1505-7B for mrc1Δ, HKY1538-1A, HKY1538-5C, and HKY1538-12A for mrc1-AQ, and HKY1323-1B and HKY1323-12D for srs2Δ.

FIG. 5.

srs2Δ interactions with replication checkpoint defective mutants. (A) Five tetrads are shown from each cross. The circles in the top panel, srs2Δ sml1-1 (HKY1369-2D) × rad53Δ sml1-1 (HKY987-2B), mark rad53Δ sml1-1 spore segregants. The squares in the top panel mark srs2Δ rad53Δ sml1-1 spore segregants. The circles in the lower panel, srs2Δ mec1Δ sml1Δ (HKY1494-7A) × mrc1Δ sml1Δ (HKY1239-26A), mark srs2Δ mrc1Δ sml1Δ spore segregants. The squares in the lower panel mark srs2Δ mrc1Δ mec1Δ sml1Δ spore segregants. (B) Five tetrads are shown from the cross srs2Δ (HKY1303-1B) × mrc1-AQ (HKY1534-3C). The circles mark mrc1-AQ segregants. The squares mark srs2Δ mrc1-AQ spore segregants. (C) Four tetrads are shown from the cross srs2Δ mad3Δ (HKY1371-7D) × mrc1Δ (HKY1235-21D). The circle marks an srs2Δ mrc1Δ spore segregant. The squares mark srs2Δ mrc1Δ mad3Δ spore segregants.

FIG. 6.

Sister chromatid cohesion assays in mrc1 mutants. (A) Sister chromatid cohesion assays were performed for three separate chromosomal locations as marked in wild-type, mrc1Δ, and srs2Δ strains. The mean percentage of two GFP dots from 200 cells is shown. Each experiment was repeated three to four times with different transformants for each genotype. The strains used were YPH1477 (wild type), Y4986 and Y4987 (mrc1Δ) and Y5168 (srs2Δ) for chromosome V, Y819 (wt), Y4989 and Y4990 (mrc1Δ), and Y5165 (srs2Δ) for chromosome IV, and YPH1444 (wild type), Y4992 and Y4993 (mrc1Δ), and Y5247 (sr2Δ) for chromosome XV. (B) Sister chromatid cohesion on chromosome V was performed on the indicated strains, counting 200 cells each time in two independent experiments and for each genotype. The strains used were YPH1477 (wild type), Y4986 and Y4987 (mrc1Δ), Y5168 (srs2Δ), Y6234 (rad51Δ), Y6344 (rad51Δ srs2Δ), Y6187 (mrc1Δ rad51Δ), Y6219 (mrc1Δ srs2Δ rad51Δ), and Y6346 and Y6355 (mrc1-AQ).

FIG. 7.

srs2Δ synthetic sickness with ctf18Δ is relieved by loss of RAD51 function. Five tetrads are shown from the cross srs2Δ rad51Δ (HKY1403-1B) × rad18Δ. The circles mark srs2Δ ctf18Δ spore segregants. The squares mark srs2Δ ctf18Δ rad51Δ spore segregants.