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. 1998 Nov 24;95(24):14278-83.
doi: 10.1073/pnas.95.24.14278.

Physical interaction between components of DNA mismatch repair and nucleotide excision repair

P Bertrand  1 , D X Tishkoff, N Filosi, R Dasgupta, R D Kolodner
Affiliations

Physical interaction between components of DNA mismatch repair and nucleotide excision repair

P Bertrand et al. Proc Natl Acad Sci U S A. .

Abstract

Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as "bait," and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes.

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Figures

Figure 1
Figure 1
Functional properties of RAD10 prey and LexA-MSH2 bait. (A) Complementation of the UV sensitivity of a rad10 strain by expression of the RAD10 prey protein. The wild-type and rad10 strains were transformed with indicated plasmids and expression of 12CA5-RAD10 protein in the strains was induced by transferring cells into synthetic drop-out (SD) Trp− medium containing galactose followed by shaking for 4 hr. Then 10-fold serial dilutions were prepared and 10 μl of different dilutions were spotted onto SD Trp− plates containing galactose, and UV sensitivity was determined. (B) Complementation of the mutator phenotype of a msh2 strain by expression of the LexA-MSH2 protein. Patches of the indicated strains on a yeast extract/peptone/dextrose plate were replica-plated to a canavanine plate to allow visualization of Canr papilliae after incubation of the plates at 30° for 2 days.
Figure 2
Figure 2
Specific interaction of MSH2 with RAD10. The indicated baits and preys were cotransformed into EGY48 along with the lacZ reporter, pSH18–34. The preys are expressed only in the presence of galactose. (A and B) Three different transformants were patched on selective plates and then replica-plated onto 5-bromo-4-chloro-indolyl β-d-galactopyranoside (X-Gal) plates with either glucose or galactose. (C) Patches of one transformant containing each indicated pair of baits and preys replica plated onto X-Gal plates containing galactose. Interaction of a bait and a prey induces β-galactosidase activity producing blue coloration.
Figure 3
Figure 3
Coimmunoprecipitation of MSH2 and RAD10. Whole-cell extracts (200 μg) were prepared from EGY48 strains expressing LexA-tagged baits (MSH2, or DmHAIRY as a negative control) and 12CA5-tagged preys (RAD10, or ScNPL3 as a negative control). The extracts (E) then were analyzed by immunoprecipitation (IP) and Western blotting. PC, proteins eluted from protein G-Sepharose used to preclear; Ab, antibody; *, proteins nonspecifically precipitated by protein-G Sepharose.
Figure 4
Figure 4
Coimmunoprecipitation of MSH2 and different NER proteins. Whole-cell extracts (200 μg) were prepared from EGY48 expressing LexA-tagged bait (MSH2) and 12CA5-tagged preys (RAD10, RAD14, RAD2, RAD25, or ScEXO1 as positive control). The extracts (E) then were analyzed by immunoprecipitation (IP) and Western blotting. PC, proteins eluted from protein G-Sepharose used to preclear; Ab, antibody; *, proteins nonspecifically precipitated by protein-G Sepharose.
Figure 5
Figure 5
Interaction of MSH2 and RAD10 in the absence of MSH3, MSH6, MLH1, and PMS1. Whole-cell extracts (200 μg) were prepared from RKY2567 and RKY2752 expressing LexA-tagged bait (MSH2) and 12CA5-tagged prey (RAD10). The extracts (E) then were analyzed by immunoprecipitation (IP) and Western blotting. PC, proteins eluted from protein G-Sepharose used to preclear; Ab, antibody; *, proteins nonspecifically precipitated by protein-G Sepharose.
Figure 6
Figure 6
Effect of an msh2 mutation on UV sensitivity of NER mutants. The indicated mutant strains were analyzed for UV sensitivity. The graphs shown indicate the % survival observed relative to no UV irradiation. No significant killing of either the wild type or the msh2 mutant strain was observed at the indicated UV dose. Three to 10 independent experiments were performed with each set of strains and one representative example is shown. The strains used were RKY2672, RKY2706 msh2, RKY2350 rad10, RKY2351 msh2 rad10, RKY2343 rad14, RKY2344 msh2 rad14, RKY2352 rad2, and RKY2353 msh2 rad2.
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