Suppression of genetic defects within the RAD6 pathway by srs2 is specific for error-free post-replication repair but not for damage-induced mutagenesis

Abstract

srs2 was isolated during a screen for mutants that could suppress the UV-sensitive phenotype of rad6 and rad18 cells. Genetic analyses led to a proposal that Srs2 acts to prevent the channeling of DNA replication-blocking lesions into homologous recombination. The phenotypes associated with srs2 indicate that the Srs2 protein acts to process lesions through RAD6-mediated post-replication repair (PRR) rather than recombination repair. The RAD6 pathway has been divided into three rather independent subpathways: two error-free (represented by RAD5 and POL30) and one error-prone (represented by REV3). In order to determine on which subpathways Srs2 acts, we performed comprehensive epistasis analyses; the experimental results indicate that the srs2 mutation completely suppresses both error-free PRR branches. Combined with UV-induced mutagenesis assays, we conclude that the Polzeta-mediated error-prone pathway is functional in the absence of Srs2; hence, Srs2 is not required for mutagenesis. Furthermore, we demonstrate that the helicase activity of Srs2 is probably required for the phenotypic suppression of error-free PRR defects. Taken together, our observations link error-free PRR to homologous recombination through the helicase activity of Srs2.

Figure 1

Sensitivity of mms2 and srs2 mutants to UV (A) and MMS (B). (Open square) DBY747 (wild-type); (filled square) WXY671 (mms2); (open circle) WXY683 (srs2); and (filled circle) WXY685 (mms2 srs2). All strains are the isogenic derivatives of DBY747. The results are an average of three independent experiments with standard deviations.

Figure 2

The effect of the srs2 mutation on the spontaneous mutation rate of mms2. Spontaneous mutagenesis assay was performed as described in the Materials and Methods. DBY747 (WT, wild-type), WXY671 (mms2), WXY683 (srs2) and WXY685 (mms2 srs2) were assayed in the same experiment. The results are an average of five independent experiments with standard deviations shown as error bars.

Figure 3

Sensitivity of rad5, pol30-46 and srs2 mutants to killing by MMS. (A) srs2 and rad5; (B) srs2 and pol30-46; (C) srs2 and pol30-46 rad5. (Square) PY39-0 (wild-type); (cross) WXY1067 (srs2); (filled circle) WXY857 (rad5); (open circle) WXY1068 (rad5 srs2); (filled triangle) PY39-46 (pol30-46); (open triangle) WXY1069 (pol30-46 srs2); (filled diamond) WXY858 (pol30-46 rad5); (X) WXY1070 (pol30-46 rad5 srs2). All the strains are isogenic derivatives of PY39-0. The results are the average of three independent experiments.

Figure 4

Sensitivity of rad30 and srs2 mutants to UV (A) and MMS (B). (Open square) DBY747 (wild-type); (filled square) WXY1071 (rad30); (open circle) WXY683 (srs2); and (filled circle) WXY1072 (rad30 srs2). All strains are the isogenic derivatives of DBY747. The results are an average of three independent experiments with standard deviations.

Figure 5

Sensitivity of rev3 and srs2 mutants to MMS-induced killing. (Open square) DBY747 (wild-type); (filled square) WXY683 (srs2); (open circle) WXY381 (rev3); and (filled circle) WXY669 (rev3 srs2). All strains are the isogenic derivatives of DBY747. The results are an average of three independent experiments.

Figure 6

Relative sensitivity of mms2, rev3 and srs2 mutants to killing by MMS in a gradient plate assay. YPD and YPD + 0.01% MMS gradient plates were made as described in the Materials and Methods. Overnight cell cultures were replicated onto each plate and incubated at 30°C for 42 h before taking photograph. Lane 1, DBY747 (wild-type); lane 2, WXY671 (mms2); lane 3, WXY382 (rev3); lane 4, WXY683 (srs2); lane 5, WXY674 (mms2 rev3); lane 6, WXY737 (mms2 rev3 srs2). All strains are the isogenic derivatives of DBY747. Arrow indicates increased MMS concentration.