The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations

Abstract

The Saccharomyces cerevisiae genome encodes four MutL homologs. Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches. We have investigated the role of MLH3 in mismatch repair. Mutations in MLH3 increased the rate of reversion of the hom3-10 allele by increasing the rate of deletion of a single T in a run of 7 Ts. Combination of mutations in MLH3 and MSH6 caused a synergistic increase in the hom3-10 reversion rate, whereas the hom3-10 reversion rate in an mlh3 msh3 double mutant was the same as in the respective single mutants. Similar results were observed when the accumulation of mutations at frameshift hot spots in the LYS2 gene was analyzed, although mutation of MLH3 did not cause the same extent of affect at every LYS2 frameshift hot spot. MLH3 interacted with MLH1 in a two-hybrid system. These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex.

Figure 1

Phylogenetic tree of MutL-related proteins. The dendrogram was generated with Megalign (DNAStar) by the clustal method with the weight table set to identities, a gap penalty of 10, and a gap length penalty of 10. All sequences used are full length and were retrieved from GenBank.

Figure 2

Spectra of mutations reverting the hom3–10 allele in wild-type and different mutant strains. Nucleotides 623–682 of the sequenced region is shown. Each independent frameshift event detected is indicated over the sequence with a Δ. Complex mutations that include base changes are shown below the sequence in parentheses. In the wild–type strain, two identical complex events were observed. The total number of isolates analyzed was: wild type, 24; msh6, 28; mlh3, 24, and mlh3 msh6, 30.

Figure 3

Spectra of mutations reverting the lys-2Bgl allele in wild-type and different mutant strains. The region comprising nucleotides 350–444 and 473–494 is shown. Each frameshift reversion is indicated on top of the sequence depicted by a Δ. Reversion events involving single basepair deletions and base changes are shown in parentheses below the sequence and insertions are indicated by a ∧. The deletion of an A occurring at position 467 in a revertant of the wild-type strain is indicated over the –. The number of revertants analyzed was: wild type, 40; msh6, 42; mlh3, 46; mlh3 msh6, 51; msh3, 46; mlh3 msh3, 41.

Figure 4

Interaction of MLH3 and MLH1 by two-hybrid analysis. Yeast strain RDKY2926 was cotransformed with the indicated combination of one bait plasmid and one prey plasmid, and the transformants were replica plated onto Ura⁻His⁻Trp⁻ 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal) glucose and Ura⁻His⁻Trp⁻ X-Gal galactose indicator plates and incubated at 30°C for 2 days.

Figure 5

Model of action of MLH3 in mismatch repair. Note that specific contacts between individual MLH and MSH proteins are not meant to be implied by this model as no information is presently available about such contacts.