(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 …
(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 …
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.
(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 …
(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 …
(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 …
(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 …
(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 …
(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.
(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 …
(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 …
(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 …
(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; …
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 …
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 …
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 |
Highest resolution shell is shown in parenthesis.
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) |
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.