Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates

Abstract

DNA mismatch repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae, mispairs are primarily detected by the Msh2-Msh6 complex and corrected following recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. We found that the Msh2-Msh6 complex is an S phase component of replication centers independent of mispaired bases; this localized pool accounted for 10%-15% of MMR in wild-type cells but was essential for MMR in the absence of Exo1. Unexpectedly, Mlh1-Pms1 formed nuclear foci that, although dependent on Msh2-Msh6 for formation, rarely colocalized with Msh2-Msh6 replication-associated foci. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These findings suggest the presence of replication machinery-coupled and -independent pathways for mispair recognition by Msh2-Msh6, which direct formation of superstoichiometric Mlh1-Pms1 foci that represent sites of active MMR.

Figure 1

Msh2-Msh6 forms nuclear foci that colocalize with replication factories. Cells expressing Msh6-mCherry (or GFP) and a DNA replication related protein tagged with 4GFP (or mCherry) were analyzed by deconvolution microscopy. (A) Images of cells with different bud size expressing Msh6-mCherry and Pol2-4GFP. (B) Distribution of Msh6-mCherry and Pol2-4GFP foci according to bud size: no bud, small (<1.5μm), medium (1.5–3μm) or large (>3μm) budded cells. Error bars indicate standard error of the mean, and “n” indicates the number of cells examined. (C) Msh6 colocalized with other components of the replisome including: Pol30, Pol3, Pol1, Rfa1 but not with the helicase subunits Mcm2 or Mcm4. Yellow boxes (2μm square) in “Bright-field” correspond to the nucleus and were enlarged (without interpolation) for the fluorescent images. White arrows indicate mCherry/GFP foci and arrowheads indicate colocalized foci on “Merge” images.

Figure 2

Msh2-Msh6 replication-associated foci depend on interactions with PCNA. (A) Cells expressing Msh6-GFP or the Msh6-F33AF34A-GFP mutant protein were analyzed for colocalization with Pol30-mCherry. (B) Quantification of the percentage of total nuclei containing Msh6-GFP or Msh6-mCherry foci for the indicated wild-type and mutant strains. (C) Images of wild-type cells or the msh6-F33AF34A mutant expressing Msh2-mCherry. (D) Quantification of the percentage of total nuclei containing Msh2-mCherry foci for the wild-type, msh3Δ or msh6-F33AF34A mutant.

Figure 3

MSH6 mutants that are unable to interact with PCNA do not complement the mutator phenotype of an msh3Δ msh6Δ exo1Δ strain. Mutation rates of msh3Δ msh6Δ (A) or msh3Δ msh6Δ exo1Δ (B) strains containing the indicated vector, MSH6 plasmid or MSH6-NTR mutant plasmids were determined by fluctuation analysis using the hom3-10 reversion assay. Rates are shown as a percentage relative to the maximum mutation rate (empty vector). * indicates p-value < 0.001 and ** indicates p-value >0.15 both relative to the empty vector rate. *** indicates p-value < 0.015 relative to the vector containing wild-type MSH6. Two-tailed p-values were determined by Mann-Whitney tests.

Figure 4

Pms1 forms foci that rarely colocalize with Msh6 foci. (A) Cells expressing Msh6-mCherry and Pms1-4GFP were analyzed by deconvolution microscopy. Representative images illustrating Pms1-4GFP and Msh6-mCherry foci that do not colocalize, do colocalize or partially colocalize. (B) Distribution of the percentage of nuclei containing Msh6-mCherry or Pms1-4GFP foci according to bud size (bud-size categories were done as in Figure 1B). (C) Quantification of the average number of Msh6 or Pms1 molecules present per focus, using the centromere protein Cse4 as a standard. (D) Time-lapse images of Pms1-4GFP were collected at the indicated intervals in a wild-type strain.

Figure 5

Pms1 foci are sites of MMR. Pms1 foci were abolished in the absence of an Msh2/Msh6 complex and increased in response to induction of mispairs or downstream MMR-recognition defects. (A) Quantification of the percentage of total nuclei containing Pms1-4GFP foci in the wild-type and indicated mutants. (B) Percentage of total nuclei containing Msh6 and/or Pms1 foci (and colocalization) for strains expressing Msh6-mCherry and Pms1-4GFP in addition to the indicated polymerase mutations. (C) Quantification of the Pms1-4GFP foci abundance for wild-type or exo1Δ, exo1Δ msh2Δ, pms1-E707K or pms1-E707K msh2Δ mutants.

Figure 6

Model of MMR pathways. Replication- and repair-associated foci are intermediates of MMR. At least two independent pathways act in preventing the accumulation of mispairs; one is coupled to the replication machinery through the interaction between Msh6 and PCNA, and acts as a sensor of potential mispairs. An alternative pathway might exist which does not require the Msh2-Msh6 association to replication factories. After mispair recognition, Msh2-Msh6 is able to recruit multiple molecules of Mlh1-Pms1 to the site of the mispair. This is followed by activation of the endonuclease activity of Pms1 and Exo1 recruitment. Inactivation of either of these two last steps might lead to an accumulation of these Pms1 foci intermediates.