Functional and physical interaction of yeast Mgs1 with PCNA: impact on RAD6-dependent DNA damage tolerance

Abstract

Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.

FIG. 1.

Loss of SUMO modification of PCNA at K164 suppresses the growth defect of mgs1Δ rad18Δ cells. Tetrads from indicated heterozygous diploids were dissected and grown on YPAD at 30°C for 3 days. (A) Squares indicate mgs1 rad18 mutants. (B and C) Circles indicate mgs1 rad5 mutants; squares indicate mgs1 rad5 rev3 and mgs1 rad5 rad30 triple mutants. (D) Squares indicate mgs1 pol30(K127R/K164R) mutants. (E, F, G, and H) mgs1 rad18 double mutants are indicated by circles. Squares indicate mgs1 rad18 pol30(K127R), mgs1 rad18 pol30(K164R), mgs1 rad18 pol30(K127R/K164R), mgs1 rad18 siz1 triple mutants.

FIG. 2.

Suppression of temperature sensitivity in mgs1-18 rad18Δ cells. (A) The siz1 or pol30(K164R) mutations suppress the temperature sensitivity for growth of the mgs1-18 rad18Δ cells. Cells were streaked onto YPAD plates and incubated at 26°C (left) or 37°C (right) for 3 days. (B) Growth of the mutant cells after temperature shift to 37°C. Cells were grown in liquid YPAD to early logarithmic phase at 26°C and then shifted to 37°C. Cells were taken at the indicated time points and plated onto YPAD plates. CFU were counted after 3 days of incubation at 26°C. (C) Detection of SUMO modification of PCNA at K164. Yeast cell extracts derived from the indicated cells were separated by SDS-PAGE, and the Western blots were probed with anti-PCNA antibody. The positions of the PCNA-SUMO conjugates and PCNA are indicated on the left. ✽, nonspecific band. (D) FACS analysis of the DNA content of synchronized cells. Cells grown to early log phase at 26°C were arrested in G₁ with α-factor for 2 h. The cells were then released into SC medium at 37°C. Aliquots were taken at the indicated time points. DNA content was measured by FACS. (E) PCNA-SUMO modification in the mgs1-18 rad18Δ strain at the restrictive temperature. Synchronized cells were released at 37°C and PCNA-SUMO conjugates were detected by Western blotting as described for panel C. α-Tubulin was used as a loading control.

FIG. 3.

Epistatic relationship between SUMO-PCNA and Srs2. (A) mgs1-18 rad18Δ siz1Δ or mgs1-18 rad18Δ srs2Δ cells were transformed with pRS425 (vector), pPCNA, and pSUMO-PCNA. Cells were streaked onto SC-Leu plates and incubated at 26°C (left) or 37°C (right) for 3 days. (B) mgs1-18 rad18Δ pol30K164R cells that carried the indicated POL30 or SUMO-fused POL30 alleles on a plasmid were streaked onto SC-Leu plates and incubated at 26°C (top panel) or 37°C (bottom panel) for 3 days. (C) MMS sensitivity of wild-type and srs2Δ cells expressing SUMO-PCNA fusion protein. For quantitative assay, cells were incubated in SC-Leu containing 0.1% MMS at 30°C. At the indicated times, samples were withdrawn and plated on medium lacking MMS. Error bars indicate the standard deviations of independent experiments. Symbols: ⧫, WT/vector; ▪, WT/pPCNA; •, WT/pSUMO-PCNA; ⋄, srs2Δ/vector; □, srs2Δ/pPCNA; ○, srs2Δ/pSUMO-PCNA. WT, wild type.

FIG. 4.

Mgs1 physically interacts with PCNA. (A) Mgs1 truncated proteins are indicated schematically on the left. Purified proteins were detected by Coomassie blue staining and are shown on the right. Lanes 1 to 6 of the gel correspond to constructs 1 to 6 shown on the left. (B) Interaction of PCNA with Mgs1. Proteins were incubated with GST (lanes G) or GST-PCNA (lanes P) for 30 min at 15°C in the presence of 0.25% NP-40 (lanes 1 to 12) or 0.02% NP-40 (lanes 13 to 16). The complexes were analyzed by SDS-PAGE and detected by silver staining. Lanes 1 and 2, wild-type Mgs1; lanes 3 and 4, Mgs1(1-128); lanes 5 and 6, Mgs1(1-386); lanes 7 and 8, Mgs1(128-386); lanes 9 and 10, Mgs1(128-588); lanes 11 and 12, Mgs (386-588); lanes 13 and 14, wild-type Mgs1; lanes 15 and 16, Mgs1(1-128). (C) His-Mgs1 or His-MgsA protein was incubated with PCNA. Protein complexes were precipitated by using Ni-NTA beads. The complexes were analyzed by SDS-PAGE and detected by silver staining. (D) Full-length and truncated versions of Mgs1 were tested for two-hybrid interaction with PCNA. Positive interactions were detected by growth on synthetic medium lacking histidine in the presence of 3-AT.

FIG. 5.

Overexpression of MGS1 inhibits Rad18 function. (A) Wild-type and rad18Δ, rad51Δ, srs2Δ, and siz1Δ mutant cells harboring empty vector (vector) or plasmids with a galactose-inducible wild-type MGS1 gene (pGal-Mgs1) were grown in liquid SC glucose-Leu or SC galactose-Leu, respectively. Cells were diluted and spotted onto SC glucose-Leu or SC galactose-Leu plates with the indicated concentrations of MMS. The plates were incubated at 30°C for 3 days. (B) Cells harboring plasmid were grown and spotted onto plates as described for panel A. DNA damage was induced by UV.