DnaN clamp zones provide a platform for spatiotemporal coupling of mismatch detection to DNA replication

Abstract

Mismatch repair (MMR) increases the fidelity of DNA replication by identifying and correcting replication errors. Processivity clamps are vital components of DNA replication and MMR, yet the mechanism and extent to which they participate in MMR remains unclear. We investigated the role of the Bacillus subtilis processivity clamp DnaN, and found that it serves as a platform for mismatch detection and coupling of repair to DNA replication. By visualizing functional MutS fluorescent fusions in vivo, we find that MutS forms foci independent of mismatch detection at sites of replication (i.e. the replisome). These MutS foci are directed to the replisome by DnaN clamp zones that aid mismatch detection by targeting the search to nascent DNA. Following mismatch detection, MutS disengages from the replisome, facilitating repair. We tested the functional importance of DnaN-mediated mismatch detection for MMR, and found that it accounts for 90% of repair. This high dependence on DnaN can be bypassed by increasing MutS concentration within the cell, indicating a secondary mode of detection in vivo whereby MutS directly finds mismatches without associating with the replisome. Overall, our results provide new insight into the mechanism by which DnaN couples mismatch recognition to DNA replication in living cells.

Figure 1. MutSF30A is unable to bind mismatches in vitro

(A) The following conserved motif necessary for mismatch binding (GXFYXXXXXDA) is found in a wide range of eukaryotic and prokaryotic MutS homologs. Indicates (*) organisms previously demonstrated that substitution of the conserved phenylalanine residue (F) eliminates mismatch binding in vitro and prevents MMR in vivo. (B) 1 μg of purified MutS and MutSF30A protein electrophoresed on a 4–15% SDS-PAGE gradient gel. (C) In vitro binding of MutS and MutSF30A to T-bulge substrate. Legend: black squares and pink diamonds show MutS and MutSF30A interaction with T-bulge containing DNA, respectively, and blue triangles show MutS interaction with homoduplex DNA. (D) An immunodot blot (far western) analysis was performed to monitor interaction between MutS or MutSF30A with DnaN. Purified MutS and MutSF30A were blotted onto a nitrocellulose membrane over a range of 0.625 pmol to 40 pmols of dimer. Purified DnaN was incubated with the membrane and probed with affinity-purified antisera against DnaN in a 1:500 dilution. Shown (left most blot) is the purified antisera control against purified MutS and MutSF30A. Shown (right most blot) is the retention of DnaN by MutS and MutSF30A as described in “Experimental Procedures.” (E) In vivo steady state levels of MutS, MutL, and DnaN. A total of 5 μg of cell extract derived from the indicated strain was electrophoresed and immunoblotted in the indicated lanes.

Figure 2. MutS-GFP forms foci independent of mismatch formation

(A) Representative images of MutS-GFP or MutSF30A-GFP foci in cells with or without 600 μg/mL of 2-AP. The white bar is 4 μm. (B) An immunoblot of the indicated MutS derivative and DnaN as a loading control. (C) Bar graph of the groups represented in (A) showing the percentage of cells with MutS-GFP foci. Total number of cells scored for each condition was from left to right: n=1234, 1410, 2380, 1222, and 1797.

Figure 3. MutS-GFP colocalizes with the replisome prior to mismatch detection

(A) Representative images of CFP-Spo0J and DnaX-YFP colocalization with the replisome. (B) Representative images of colocalization between MutS-YFP with DnaX-CFP following 2-AP treatment. White arrows denote MutS-YFP foci that colocalize with the DnaX-CFP, whereas red arrows denote MutS-YFP foci that do not colocalize with DnaX-CFP (C) Representative images of colocalization between MutSF30A-YFP with DnaX-CFP following 2-AP treatment. The vital membrane stain TMA-DPH is shown in blue, the white bar is 4 μm. (D) Scoring of colocalization of MutS-YFP and MutL-GFP at the replisome in the presence and absence of 2-AP, p-values are one- tailed; p= * 2.03×10⁻⁵; **2.77; ***4.77 × 10^{−10; #} 0.0568

Figure 4. Elevated expression of MutS-GFP increases replisome-associated foci

(A) Elevated expression of mutS-gfp and mutSF30A-gfp increases the percentage of cells with MutS foci. The first four groups contain a ΔmutS deletion and the expressed GFP fusion represents the sole copy of mutS within the cell. The second four groups represent a deletion of the entire mutSL locus with mutS- or mutSF30A-GFP expressed ectopically from a P_spac promoter (B) Elevated expression of mutS-gfp causes an increase in colocalization of MutS foci to DnaX-mCherry foci. Total number of cells scored is 106–196. (C) MutSF30A-GFP foci expressed from the mutS native locus in the dnaN5 background at 30 and 37 °C.

Figure 5. Mismatch detection by MutS800-GFP induces focus formation at nascent DNA when ectopically expressed

(A) MutS800-GFP foci form in response to mismatches independent of DnaN (faint foci indicated by white circles) (B) MutSF30A800 fails to form foci. The vital membrane stain TMA-DPH is shown in red and the white scale bar is 4 μm. (C) Bar graph of ectopically expressed MutS800-GFP and MutSF30A800-GFP foci with and without 2AP. From left to right, total cells scored: 1114, 1154, 883, 1343. P-values are 1-tailed: *p=2.69×10⁻¹⁷, **p=1.20×10⁻⁵, ***p=3.22×10⁻⁷. (D) Bar graph of ectopically expressed MutS800-GFP foci within the dnaN5 background revealed no statistical difference at 30 °C and 37°C (p=0.38).

Figure 6. DnaN-independent focus formation of MutS800-GFP localizes to the same subcellular position as MutS-YFP

(A) Position of MutS-GFP, ectopically expressed MutS800-GFP, and DnaX-GFP foci scored relative to cell length (n=125 cells for each group). (B) 29.9+6.3% (n=204) of the MutS800-GFP colocalizes with DnaX-mCherry. These results are not statistically different with p=0.099 when compared with MutS-YFP colocalization with DnaX-CFP 35.5+5.6 shown in Figure 3. The left most image is the negative image, MutS800-GFP, DnaX-mCherry, and a merge. The membrane is stained with TMA-DPH and is shown in blue, and the scale bar represents 4 μm. (C) We measured the inter-focal distance (IFD) between MutS-YFP and MutS800-GFP foci that failed to colocalize with DnaX-mCherry foci. No IFDs were measured less than 0.2 μm. We measured 94 MutS800-GFP, DnaX-mCherry and 105 MutS-YFP, DnaX-CFP focal pairs that failed to colocalize. Each bar represents the percentage of cells with IFD between the indicated distance.

Figure 7. Model for temporal coupling of MutS to DNA replication

MutS relies on a DnaN clamp zone- to target MutS to nascent DNA. DnaN-dependent mode of mismatch detection represents 90% of repair. This figure is adapted from the clamp zone model (Su’etsugu & Errington).