MutS/MutL crystal structure reveals that the MutS sliding clamp loads MutL onto DNA
- Netherlands Cancer Institute, Netherlands
- Justus-Liebig-University, Germany
- Erasmus Medical Center, Netherlands
- MRC Laboratory of Molecular Biology, United Kingdom
Figures
Figure 1 with 2 supplements
Crystal structure of the crosslinked MutSΔC800/MutLLN40 complex.
(A) DNA and ATP-dependent crosslinking of MutSΔC800 D246C (S) and MutLLN40 N131C (L) and large-scale purification. Constructs and domain definitions are shown. (B) Crystal structure of the trapped …
Figure 1—figure supplement 1
Crosslinking, purification and crystal structure of the 856 MutSΔC800/MutLLN40 complex.
(A) Single-cysteine MutS D246C and single-cysteine MutL 857 N131C constructs with replaced and introduced cysteine positions are shown, and colored 858 according to domain definitions in main text …
Figure 1—figure supplement 2
Electron density for different crystal forms of the MutSΔC800/MutLLN40 complex.
Electron density shown in region around the domain indicated at contour level 1.0 rmsd and 3.50 rmsd for the difference density map. (A) Crystal form 1, (B) Crystal form 2, (C) Crystal form 3.
Figure 2 with 2 supplements
The structure of the MutSΔC800/MutLLN40 complex reveals the MutS sliding clamp conformation.
(A) FRET within MutS dimers (normalized for unbound protein) reveals residues 449 coming closer together upon ATP addition. Error bars depict mean ± SD, n = 3. (B) FRET assay agrees with residue 246 …
Figure 2—figure supplement 1
ATP-analog and DNA in the crystal structure.
(A) Difference density map for AMP-PNP (mFo-DFc at 2.8σ) after refinement without the nucleotide is shown for MutS subunit A in the 4.7 Å crystal structure. (B) The asymmetric unit in the P21 …
Figure 2—figure supplement 2
FRET assay – controls and raw data.
(A) FRET (normalized for unbound protein) within MutS D835R heterodimers (449-AF488/449-AF594) reveals residues 449 coming closer together upon ATP addition when bound to mismatch DNA. Bars depict …
Figure 3 with 2 supplements
Interaction of the MutSΔC800 sliding clamp with MutLLN40.
(A) Crosslinking occurs between MutSΔC800 A336C and MutLLN40 T218C using BMOE (right panel), as suggested by the structure (left panel). (B) Spontaneous mutation rates after complementing MutS or …
Figure 3—figure supplement 1
MutS–MutL interaction.
(A) MutSΔC800 D246C and MutLLN40 T218C do not crosslink efficiently with either a short (BMOE, 8 Å) or a long (BM[PEO]3, 18 Å) crosslinker, as e.g. seen by lack of MutS and MutL depletion. (B) MutS …
Figure 3—figure supplement 2
(A) MutLLN40 (L) coelutes with crosslinked MutSΔC800/MutLLN40 complex (SL) from size-exclusion chromatography (right), after incubation with 100-bp DNA with a G:T mismatch and AMP-PNP, indicating that MutL can still dimerize in this complex.
(B) Model for MSH2/MSH6 interaction with MLH1/PMS2, in which the N-terminus of MLH1 simultaneously binds to the connector domain of MSH2 and the APTase and core domains of MSH6.
Figure 4 with 1 supplement
The MutS sliding clamp positions MutL onto DNA.
(A) Model of DNA binding by the MutSΔC800/MutLLN40 complex. Three arginines in the MutLLN40 DNA-binding groove are shown as red spheres. (B) In the presence of ATP, MutSΔC800 has a fast off-rate …
Figure 4—figure supplement 1
DNA binding by the MutSΔC800/MutLLN40 complex.
(A) The model for MutSΔC800/MutLLN40 complex on DNA (orange) sterically allows for LN40 dimerization (dimer modeled in green and grey as present in pdb entry 1NHJ). (B) Analysis as in Figure 4B, but …
Figure 5 with 1 supplement
Implications for DNA mismatch repair initiation.
(A) Stopped-flow FRET and FP assay shows kinking of 45-bp DNA by MutSΔC800 binding only if there is a mismatch. Magnitude of FRET events are indicated by stars in the cartoon. (B) While MutSΔC800 …
Figure 5—figure supplement 1
DNA kinking by MutSΔC800 and MutSΔC800/MutLLN40.
(A) Stopped-flow FRET and FP assay shows kinking of 45-bp DNA by MutS binding only if there is a mismatch. Separate traces for the fluorophores are shown (orange: acceptor; green: donor fluorophore; …
Videos
Video 1
Interpolation between two MutS conformations.
Interpolation between the mismatch-bound conformation of MutS and the conformation as observed in complex with MutLLN40 shows tilting of the MutS subunits across each other. The connector domain …
Video 2
Model for initiation of DNA mismatch repair.
After MutS (cyan/blue) has recognized a mismatch in DNA (in orange; mismatch shown as pink spheres), it will bind ATP which triggers a conformational change in which the subunits tilt across each …
Tables
Table 1
Data collection and refinement statistics
| Crystal form 1 27-bp DNA | Crystal form 2 27-bp DNA | Crystal form 3 100-bp DNA | |
|---|---|---|---|
| Data collection | |||
| Space group | C2 | C2 | P21 |
| Cell dimensions | |||
| a, b, c (Å) | 165.9, 188.5, 200.4 | 380.6, 126.5, 243.3 | 192.6, 109.4, 277.5 |
| α, β, γ (°) | 90.0, 94.8, 90.0 | 90.0, 91.4, 90.0 | 90.0, 90.0, 90.0 |
| Resolution (Å)* | 82.7–4.71 (4.96–4.71) | 49.94–6.6 (7.13–6.6) | 49.3–7.6 (8.5–7.6) |
| Rmerge | 19.4 (79.7) | 21.3 (80.1) | 16.8 (91.9) |
| I/σI | 2.5 (1.0) | 3.4 (1.1) | 4.3 (1.0) |
| Completeness (%) | 97.3 (98.0) | 96.8 (97.7) | 81.3 (82.5) |
| Redundancy | 2.4 (2.4) | 2.9 (3.0) | 2.3 (2.2) |
| Refinement | |||
| Resolution (Å) | 4.7 | 6.6 | 7.6 |
| No. reflections | 31,052 | 21,305 | 11,763 |
| Rwork/ Rfree | 31.8/35.0 | 25.6/28.7 | 26.2/30.5 |
| No. atoms | 21,906 | 45,054 | 45,054 |
| Protein | 21,813 | 44,868 | 44,868 |
| Ligand/ion | 93 | 186 | 186 |
| Water | 0 | 0 | 0 |
| B-factors | |||
| Protein | 212 | 255 | 221 |
| Ligand/ion | 220 | 212 | 171 |
| Water | n/a | n/a | n/a |
| R.m.s deviations | |||
| Bond lengths (Å) | 0.009 | 0.0103 | 0.0113 |
| r.m.s. Z (bonds) | 0.45 | 0.51 | 0.55 |
| Bond angles (°) | 1.32 | 1.35 | 1.31 |
| r.m.s. Z (angles) | 0.59 | 0.70 | 0.68 |
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*
Highest resolution shell is shown in parenthesis.
Table 2
Mutation rates for MutS and MutL mutants as determined using in vivo complementation assays
| Protein | Mutations per 107 | (95% confidence interval) |
|---|---|---|
| MutS variant (MutL interface) | ||
| Empty vector | 0.601 | (0.446–0.772) |
| WT MutS | 0.0686 | (0.0408–0.101) |
| MutS P595A/I597A | 0.0545 | (0.0310–0.0826) |
| MutS M759D | 0.0819 | (0.0490–0.121) |
| MutS Y563A | 0.0488 | (0.0272–0.0749) |
| MutS P595A/I597A/M759D | 0.704 | (0.556–0.864) |
| MutS Y563A/P595A/I597A | 0.317 | (0.233–0.411) |
| MutS Y563A/P595A/I597A/M759D | 0.773 | (0.618–0.941) |
| MutL variant (MutS interface) | ||
| Empty vector | 5.43 | (4.00–7.00) |
| WT His-MutL | 0.121 | (0.0542–0.206) |
| His-MutL A138E | 2.76 | (2.12–3.46) |
| His-MutL H139A | 0.103 | (0.0439–0.179) |
| His-MutL A138E/H139A | 4.87 | (3.55–6.33) |
| His-MutL R55D/R57D | 6.41 | (4.99–7.95) |
| His-MutL R200D | 0.663 | (0.432–0.932) |
| His-MutL R55D/R57D/H139A | 5.33 | (3.93–6.89) |
| His-MutL R55D/R57D/A138E/H139A | 6.13 | (4.58–7.84) |
| His-MutL R55D/R57D/H139A/R200D | 5.22 | (3.84–6.76) |
| His-MutL R55D/R57D/A138E/H139A/R200D | 5.48 | (4.04–7.06) |
| MutL variant (DNA binding) | ||
| His-MutL R266E | 5.87 | (4.78–7.04) |
| His-MutL R162E/R266E/R316E | 5.39 | (4.37–6.49) |
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Mutation rates and 95% confidence intervals were determined using the Fluctuation AnaLysis CalculatOR (http://www.mitochondria.org/protocols/FALCOR.html) using the MSS-MLE method. For MutS, at least 24 independent colonies were picked; for MutL at least 12 independent colonies were picked.
