Single-molecule motions and interactions in live cells reveal target search dynamics in mismatch repair

Abstract

MutS is responsible for initiating the correction of DNA replication errors. To understand how MutS searches for and identifies rare base-pair mismatches, we characterized the dynamic movement of MutS and the replisome in real time using superresolution microscopy and single-molecule tracking in living cells. We report that MutS dynamics are heterogeneous in cells, with one MutS population exploring the nucleoid rapidly, while another MutS population moves to and transiently dwells at the replisome region, even in the absence of appreciable mismatch formation. Analysis of MutS motion shows that the speed of MutS is correlated with its separation distance from the replisome and that MutS motion slows when it enters the replisome region. We also show that mismatch detection increases MutS speed, supporting the model for MutS sliding clamp formation after mismatch recognition. Using variants of MutS and the replication processivity clamp to impair mismatch repair, we find that MutS dynamically moves to and from the replisome before mismatch binding to scan for errors. Furthermore, a block to DNA synthesis shows that MutS is only capable of binding mismatches near the replisome. It is well-established that MutS engages in an ATPase cycle, which is necessary for signaling downstream events. We show that a variant of MutS with a nucleotide binding defect is no longer capable of dynamic movement to and from the replisome, showing that proper nucleotide binding is critical for MutS to localize to the replisome in vivo. Our results provide mechanistic insight into the trafficking and movement of MutS in live cells as it searches for mismatches.

Keywords: Bacillus subtilis; DNA replication; bacterial cell biology; single-cell analysis; super-resolution microscopy.

Fig. 1.

Location and dynamics of MutS in live B. subtilis. (A) Labeling scheme for MutS-PAmCherry. RBS, ribosome binding site. (B) Representative frames showing the photoactivation of a single copy of MutS-PAmCherry in a cell. Purple and green lines above the frames correspond to the photoactivation laser pulse and the imaging laser. (C, Lower Left) Photoactivated localization microscopy (PALM) reconstruction and (C, Right) single-molecule trajectories of MutS-PAmCherry in (C, Upper Left) a live B. subtilis cell. Each subdiffraction-limited coordinate of MutS-PAmCherry is plotted in the PALM image as a Gaussian blur with width equal to its localization uncertainty. The red arrow indicates a region of MutS accumulation. White dashed lines indicate the computer-detected cell boundary. (Scale bars: 1 μm.)

Fig. 2.

Positioning of the replisome. (A) Sample fluorescence image of DnaX-mCitrine in 5 cells and localization probability of DnaX in 161 cells along the longitudinal cell axis (Inset). The replisome appears most frequently at the one-quarter positions in exponential-phase cells. (B) Photobleaching-assisted localization of single DnaX-mCitrine molecules within a cluster. The intensity of a cluster is plotted against time (20 ms per frame), and photobleaching events are identified by maximum likelihood estimation. The point spread function (PSF) images of photobleached single molecules can then be obtained by subtracting the average intensity of the frames after the photobleaching from that of preceding frames (schematic representation shown in Inset), allowing the precise location of the photobleached molecule to be determined through 2D Gaussian fitting. Representative PSFs for two photobleached mCitrine molecules are shown below the cluster intensity trajectory. (C) Distribution of separation distance between DnaX-mCitrine within a replisome as determined from photobleaching-assisted localization, with two sample overlapping PSFs shown. (D, Left) The radii explored by DnaX-mCitrine as calculated by tracking the motion of each cluster centroid using low-power time-lapse imaging and (D, Right) the size distribution of the domain explored by each cluster, illustrating the subtle replisome motion that explores small domains of ∼84 nm in radius on average. (Scale bar: 1 μm.)

Fig. 3.

Two-color imaging results from single cells expressing both MutS-PAmCherry and DnaX-mCitrine. (A) Photoactivated localization microscopy reconstruction of MutS-PAmCherry (magenta) in a cell (Top) with MutS-enriched regions indicated with white arrowheads and (Middle) overlaid with DnaX-mCitrine clusters (green). A representative time-coded trajectory showing MutS entering, dwelling at, and leaving one of the replisome regions is shown in Bottom. (Scale bars: 1 μm.) (B, Upper) Separation distance from the replisome and (B, Lower) instantaneous speed as a function of time for the MutS trajectory shown in A. Gray indicates the time spent in the replisome region. The red curve indicates a prolonged period of decreased MutS speed. Black dashed lines indicate (Upper) 100-nm MutS-DnaX separation distance and (Lower) average DnaX speed (0.5 μm/s as measured by tracking cluster centroids). (C) Cross-correlation between the separation between MutS and the center of DnaX cluster and the instantaneous speed of MutS from 29 trajectories in 11 cells normalized from −1 to 1 (66). Error bars represent SEM. (D) Cumulative probability distribution of the time period that MutS (red “S”) spends within the same replisome region (blue “R”) fit to a two-term exponential decay function (dashed line) P = A₁exp(−t/τ₁) + A₂exp(−t/τ₂), where A₁ = 0.42, A₂ = 0.58, τ₁ = 25 ms, and τ₂ = 188 ms. The error bars are SDs from seven rounds of bootstrapping. The dwell time distribution is constructed using 751 trajectories at least 10 frames long for molecules that can be tracked from the time that they enter the replisome region until they leave the replisome (Inset).

Fig. 4.

MutS localization and dynamics in WT cells. (A) Photoactivated localization microscopy (PALM) reconstruction (magenta) and single-molecule trajectories (red) of MutS-PAmCherry overlaid with DnaX-mCitrine (green and blue) and phase-contrast cell images. Overlapping signals are colored in white. Orange arrows indicate replisome regions at which preferential MutS enrichment or dwelling is observed. (Scale bar: 1 μm.) (B and C) Localization probability density maps of (Upper) DnaX-mCitrine (blue-green) and (Lower) MutS-PAmCherry (red-yellow) within a normalized cell. White lines designate the one-quarter, one-half, and three-quarters positions along the cell long axis and the one-half position in the transverse direction. In total, (B) 108 WT− and (C) 91 WT+ cells with two replisome clusters were used to generate the corresponding density maps. To allow for quantitative comparison of colocalization between different cases, the Pearson correlation coefficients between each pair of DnaX/MutS density maps were calculated. The correlation coefficients for WT− and WT+ cells are 0.83 and 0.81, respectively. Grid pixel size: ∼100–200 nm. (D) Diffusion coefficients of MutS-PAmCherry as a function of separation distance from the nearest replisome. Error bars indicate 95% confidence interval. The three arrows point to the 50-, 200-, and 400-nm separation distances (from left to right) at which the MutS diffusion coefficient distributions are further analyzed in F. (E) Distribution of the probability of a mismatch occurring in an observed cell under normal growth condition (blue) and 2-AP treatment (red) over time. The vertical purple dashed line indicates the average duration (210 s) of observation for each cell in PALM experiments. (F) Distribution of the MutS diffusion coefficients at three distances away from the replisome. Over 3,000 trajectories were analyzed for both WT− and WT+ cells.

Fig. 5.

Response of MutS to sequential blocking of MMR steps. (A) Schematic diagrams showing the first four steps of MMR, including replisome binding, mismatch recognition, ATPase activity, and MutL recruitment, each of which is blocked in one of four mutant strains. (B, F, and J) Two-color images of representative cells from MutS800, MutS[F30A], and MutS[K608M] strains. (C, G, K, and N) Localization probability density maps of untreated cells for each strain generated from N cells with two replisome clusters. Pearson correlation coefficients between DnaX and MutS densities are listed in the lower right corner of corresponding MutS density maps. Note that MutS density maps may exhibit similar intensity levels but have different correlation coefficients because of differences in the positioning of corresponding DnaX density maps. (D, H, L, and O) Density maps of 2-AP–treated cells. (P) Density maps for 2-AP/HPUra double-treated cells from the ΔmutL+ strain. HPUra restores MutS enrichment around the replisome and also causes simultaneous shifting of DnaX and MutS localizations toward cell center. (E, I, M, and Q) Diffusion coefficients of MutS-PAmCherry variants as a function of separation distance from the nearest replisome. Error bars indicate 95% confidence interval.

Fig. 6.

MutS recruitment to DNA on replication initiation and interaction with DNA polymerase subunits. (A) Analysis of pooled ChIP-seq data from two independent experiments showing the enrichment levels of MutS and the polymerases DnaE and PolC along the chromosome 10 min after DNA replication initiation. Pearson correlation coefficients (r values) are shown for genome-wide ChIP-seq enrichment profiles of DnaE/MutS and PolC/MutS. The position of oriC is indicated by an arrow above the plots. (B) Overlaid enrichments of MutS, PolC, and DnaE from A in a window of the genome near oriC. (C) Co-IP of DnaE and PolC with MutS using affinity-purified antiserum directed against MutS. Lane 1, 5% input; lane 2, anti-MutS immunoprecipitation.