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. 2013 Oct;9(10):e1003920.
doi: 10.1371/journal.pgen.1003920. Epub 2013 Oct 31.

Different roles of eukaryotic MutS and MutL complexes in repair of small insertion and deletion loops in yeast

Nina V Romanova  1 , Gray F Crouse
Affiliations

Different roles of eukaryotic MutS and MutL complexes in repair of small insertion and deletion loops in yeast

Nina V Romanova et al. PLoS Genet. 2013 Oct.

Abstract

DNA mismatch repair greatly increases genome fidelity by recognizing and removing replication errors. In order to understand how this fidelity is maintained, it is important to uncover the relative specificities of the different components of mismatch repair. There are two major mispair recognition complexes in eukaryotes that are homologues of bacterial MutS proteins, MutSα and MutSβ, with MutSα recognizing base-base mismatches and small loop mispairs and MutSβ recognizing larger loop mispairs. Upon recognition of a mispair, the MutS complexes then interact with homologues of the bacterial MutL protein. Loops formed on the primer strand during replication lead to insertion mutations, whereas loops on the template strand lead to deletions. We show here in yeast, using oligonucleotide transformation, that MutSα has a strong bias toward repair of insertion loops, while MutSβ has an even stronger bias toward repair of deletion loops. Our results suggest that this bias in repair is due to the different interactions of the MutS complexes with the MutL complexes. Two mutants of MutLα, pms1-G882E and pms1-H888R, repair deletion mispairs but not insertion mispairs. Moreover, we find that a different MutL complex, MutLγ, is extremely important, but not sufficient, for deletion repair in the presence of either MutLα mutation. MutSβ is present in many eukaryotic organisms, but not in prokaryotes. We suggest that the biased repair of deletion mispairs may reflect a critical eukaryotic function of MutSβ in mismatch repair.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. An assay for loop repair.
(A) The initial strains used to construct the assay for in/del loop repair were a set of isogenic strains containing the LYS2 gene replacing the HIS4 gene near the ARS306 origin of replication as shown above . “Same” and “Opposite” refer to the orientation of the LYS2 gene relative to the orientation of the original HIS4 gene . The wild-type LYS2 sequences were subsequently replaced with sequences to create either the −1 frameshift allele lys2ΔA746 or the +1 frameshift allele lys2ΔBgl –. (B) The −1 lys2ΔA746 and the +1 lys2ΔBgl frameshift alleles can be reverted to wild-type by a compensating addition or deletion of nucleotides anywhere within an approximately 200-bp reversion window –. Oligos with sequences corresponding to two different locations within the reversion window of the mutant alleles and ranging in size from 31–36 nt were used to produce Lys+ revertants (Table S4). The colors indicated are those used in subsequent figures and also in Table S4. The red and yellow oligos induce a 2-nt loss in lys2ΔA746 strains and the blue and green oligos insert 2 nt into lys2ΔBgl strains. Single-stranded oligos are used for transformation, and can therefore have the sequence of either the transcribed (Tr) or non-transcribed strand (NTr). Oligos inducing 1-nt in/del mutations follow a similar color and naming scheme (see text for details). (C–F) Oligos transform by serving as primers for subsequent replication, on either the leading or lagging strands of replication. If the mismatch created by the oligo is not removed during replication, a reverting frameshift will result in the next round of replication. Additional nucleotides in the oligo will create a primer-strand loop and thus an insertion mutation; missing nucleotides in the oligo will create a loop on the template strand and thus lead to a deletion mutation. (C) and (D) indicate that the same oligo (an oligo with the sequence of the transcribed strand (Tr) in location 2 (L2) adding a sequence of TC) will anneal to the leading strand of replication in lys2ΔBgl strains of the Opposite orientation (TrL2-lead-o) or to the lagging strand in strains of the Same orientation (TrL2-lag-s). (E) and (F) show the same process for an oligo inducing a deletion of GA in lys2ΔA746 strains by annealing on the leading strand in strains of the Opposite orientation (TrL2-lead-o) or on the lagging strand of strains in the Same orientation (TrL2-lag-s).
Figure 2
Figure 2. Effect of MMR on 2-nt in/del mismatches.
The mean number of Lys+ revertants, with standard deviation, is shown for the indicated oligo and strain combination. The coloring is explained in Figure 1 and oligo sequences are given in Table S4. TrL1 and TrL2 refer to oligos with the sequence of the transcribed strand in Location 1 and 2, respectively. For the Tr oligos, annealing to the lagging strand occurs in strains with the Same orientation (Lag-s). The fewer transformants obtained for a given oligo and strain combination, the better the repair for the mismatch created by the oligo. Oligos creating insertion loops are transformed into lys2ΔBgl strains and oligos creating deletion loops are transformed into lys2ΔA746 strains. As an example, all TrL1 oligos are essentially identical in sequence, with the exception that the “blue” oligo inserts a +GA loop, the “green” oligo inserts a +TC loop, and the “red” oligo causes a 2-nt −GA deletion loop in the template strand opposite the location of the + loops in the other two oligos. There is no active MMR in msh2 strains, whereas msh3 strains have MutSα present and msh6 strains contain MutSβ.
Figure 3
Figure 3. The effect of mutations in PMS1 on 2-nt in/del mispairs.
Oligos were transformed into strains of the indicated genotypes and analyzed as in Figure 2; the msh2 results are those given in Figure 2.
Figure 4
Figure 4. Effect of MMR on 1-nt in/del mismatches.
TrL1 Oligos were transformed into Same-orientation strains of the indicated genotypes and analyzed as in Figure 2 (TrL1-Lag-s). For 1-nt in/del mismatches, oligos creating insertion loops are transformed into lys2ΔA746 strains and oligos creating deletion loops are transformed into lys2ΔBgl strains. Only MutSβ is present in msh6 strains and only MutSα is present in msh3 strains.
Figure 5
Figure 5. Effect of Mlh3 on 2-nt deletion mispairs.
Oligos were transformed into strains of the indicated genotypes and analyzed as in Figure 2. (Data for msh2, msh6, and pms1-H888R are from Figures 2 and 3.)
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