1. The N Terminus of Saccharomyces cerevisiae Msh6 Is an Unstructured Tether to PCNA 
Scarlet S. Shell, Christopher D. Putnam, Richard D. Kolodner 
Molecular Cell 
Volume 26, Issue 4, Pages (May 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Partial Proteolysis of Msh2-Msh6 and the Msh6 NTR
Increasing amounts of trypsin were incubated with 1 μg purified Msh2-Msh6 or Msh6 NTR and analyzed by SDS-PAGE and silver staining or western blotting with antibodies against Msh2 or Msh6. Both Msh6 in the Msh2-Msh6 complex and the isolated Msh6 NTR were sensitive to levels of exposure to trypsin that did not digest Msh2. The upper arrow points to the band corresponding to the truncation of the Msh6 NTR, whereas the lower arrow points to the band corresponding to the truncation of both the Msh6 NTR and the Msh6 MBD.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
Size Exclusion Chromatography of Msh6 NTR, PCNA, and Their Complex
(A) The Msh6 NTR eluted from a Superose 6 column more rapidly than did PCNA trimers, indicating that the NTR either oligomerizes or has an extended structure.
(B) Fifteen micrograms of PCNA was mixed with various ratios of NTR monomers, which resulted in the formation of complexes that migrated at three separate positions. A PCNA to NTR ratio of 3:1 or 3:2 gave a peak at the first position with some free NTR. Ratios of 3:2 and 3:3 gave a peak at the second position with some components at the first position as well, and ratios of 3:4.5, 3:6, and 3:9 gave a third peak with increasing amounts of unbound NTR.
(C) Heating PCNA at 100°C for 10 min caused the protein to form a high molecular weight aggregate.
(D) Similar heat treatment of the NTR did not change its elution profile.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3 SAXS of Msh6 NTR, PCNA, and Their Complex
(A) Scattering curves for 10 mg/mL Msh6 NTR, PCNA, and the PCNA-MSH6 NTR complex at a 3:2 molar ratio of monomers. The NTR and complex curves were scaled by 100 and to eliminate overlap. The solid line through PCNA represents the fitting of the S. cerevisiae PCNA trimer (PDB ID 1PLQ; Krishna et al. [1994]) to the experimental data with the program CRYSOL (Svergun et al., 1995), and the dashed line represents the fit corresponding to the ab initio PCNA model in (F).
(B) Rg of PCNA, NTR, and the PCNA-NTR complex was calculated at various concentrations with average values of 34.1 ± 0.1 Å, 56 ± 2 Å, and 70.2 ± 0.4 Å, respectively, shown by horizontal lines. The linear relationship of concentration to I(0) and consistency of Rg indicate the samples did not aggregate or dissociate during the experiment.
(C) Kratky plots of the samples show that, unlike the PCNA-containing samples, the NTR shows no globular fold.
(D) Plot illustrating the fits of the functional form for an unfolded polymer (Equation 1, Experimental Procedures) to the experimental scattering curves for 2.5, 5, and 10 mg/mL NTR. The global parameters used for all of the curves are L = 1078 Å, b = 18.7 Å, Rc = 6.99 Å . I(0) was fit independently for each curve and was found to be 461, 242, and 106 intensity units for the 10, 5, and 2.5 mg/mL samples, respectively.
(E) P(r)s for PCNA, NTR, and the PCNA-NTR complex.
(F) PCNA and NTR scattering curves were fitted with GASBOR (Svergun et al., 2001). Representative solutions from multiple runs are displayed as spheres at the positions of the “dummy residues” from the fitting. The reconstruction of PCNA (blue) was robust, whereas the shape of the extended NTR polypeptide (red) varied dramatically from run to run.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
Partial Proteolysis of the NTR and Full-Length Msh2-Msh6 Complexed to PCNA and Solution Structure of the Msh2-Msh6 Complex with PCNA
(A) Tryptic digestion of Msh6 NTR was essentially unaffected by the addition of PCNA to the reaction at a 1:1 monomer ratio when compared to a BSA control as observed in Coomassie blue-stained gels and western blots using antibodies against Msh6.
(B) Similarly, proteolysis of Msh6 in the Msh2-Msh6 heterodimer showed no substantial change when PCNA was added at a 1:1 monomer ratio as compared to the BSA control.
(C) Pair distribution functions for PCNA, Msh2-Msh6, and the ternary complex at 3 mg/ml Msh2-Msh6 and equimolar PCNA trimers.
(D) Pair distribution functions for 3 mg/ml Msh2-Msh6Δ or Msh2-Msh6Δ F33F34AA with and without equimolar PCNA trimers.
(E) P(r) functions calculated for randomly generated models of MutS dimers linked to PCNA via random peptides reveals that no single conformer can account for the observed P(r) curve of the Msh2-Msh6-PCNA complex.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5 Disorder Predictions for PIP Box-Containing Proteins
(A) IUPRED long-range disorder predictions for S. cerevisiae Msh2, which lacks an extended NTR region, Msh6, and Msh3. Black boxes indicate the positions of the PIP box consensus. Note that the disorder tendency parameter is substantially lower in the regions with homology to Msh2 and MutS.
(B) Averaged disorder tendency for consensus PIP-box residues and ten residues on either side for all S. cerevisiae proteins containing a PIP box consensus. Individual proteins are plotted as points in groups based on whether they bind PCNA, do not bind PCNA, or are not known to bind PCNA. Horizontal lines represent the median disorder tendency.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions