Structural and sequence analysis of MSH3.

<p>A: Multiple sequence alignment of MSH3. Jalview created visualization <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Clamp1" target="_blank">[120]</a> using the first 500 amino acids of the mouse B6 MSH3 (NP_034959.2). Conservation values and consensus sequence are based on alignment of <i>S. cerevisiae</i> Msh3p, <i>E. coli</i> MutS and 17 mammalian MSH3 homologs; values range from 0–9, where 0 is lowest and 9 is the highest. Protein interacting domains indicated pertain to those regions of the human MSH3 protein. This panel only shows an abbreviated set of the species of MSH3 sequence, the full set analysed is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280.s005" target="_blank">Figure S5</a>. B: MSH3 variant within β-turn. The T321I variant occurs within a Type I β-turn, as determined by specific backbone turn angles <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Kabsch1" target="_blank">[117]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Venkatachalam1" target="_blank">[118]</a> from the human MSH3 structure (3THW_B). Top left: hMSH3 tube diagram of Cα atoms of β-turn (blue), <i>i</i>+2 (T) residue (red) and additional three residues on N- and C-terminal ends (green). Bottom left table shows the β-turn propensity is relatively strong throughout MutS/MSH3 homologs, while the CBy variant (Isoleucine at <i>i+2</i> position) is extremely disfavored (table bottom left) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Hutchinson1" target="_blank">[69]</a>. Right: Ball and stick diagram of contact sites of Asp (D) and Thr (T) residues in β-turn with residues 194 and 214 respectively. Line diagram of Thr (T) hydroxyl group contact with neighbouring Threonine residue at position 365. The absence of the Threonine hydroxyl group may be important to stabilizing the β-turn itself, and/or may change the conformation of the turn, potentially disrupting distant contacts important for proper protein folding. MSH3 visualizations created using PyMol (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).</p

Representative CAG repeat distributions from reciprocal <i>Msh3</i> congenic lines of mice.

<p>Typical GeneScan traces for sizing of the CAG repeat as outlined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen-1003280-g001" target="_blank">Figure 1B</a>. Liver (A) and Striatum (B) from 16–20 week old R6/1 transgenic mice showing the effect of homozygosity at the <i>Msh3</i> locus on the pattern of expansion in the reciprocal congenic mice. Regardless of genetic background, CBy homozygosity at the congenic locus results in loss of somatic expansion, while B6 homozygosity is permissive of somatic expansion.</p

MSH3 coding polymorphisms and protein expression in different mouse strains.

<p>A) <i>Msh3</i> polymorphisms in <i>Msh3</i> gene from C57BL/6 (B6) and BALB/cBy (CBy) mice. Promoters were identical. SNPs were identified or confirmed to those in <i>dbSNP</i> by sequencing the <i>Msh3</i> gene. In DBA/2J, exon 8, AA#392 was correctly identified to be T/Valine. For a given amino acid the same codon was used for the variants. The complete set of MSH3 protein polymorphisms in 14 mouse strains is in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280.s009" target="_blank">Table S2</a>. B) MSH3 expression in spleen extract from different background using two different MSH3 antibodies <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Holt1" target="_blank">[65]</a>. The faster migrating band for 5A5 was a non-specific cross-reacting product, as described for 5A5 but not 2F11 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Holt1" target="_blank">[65]</a>. All other figures in this study used 2F11. C) Typical GeneScan traces for sizing of the CAG repeat as outlined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen-1003280-g001" target="_blank">Figure 1B</a>. Representative CAG repeat distributions from liver of F1 progeny between CBy and other inbred strains of mice. The top, bottom and second panel show the controls CBy (stable), B6 (unstable), and CBy X B6 (intermediate) CAG profiles, respectively. Note: Western blot data comes from inbred mice. The higher levels of MSH3 in C3H and B6 are halved in the cross to CBy.</p

Representative CAG repeat distributions, and <i>Msh3</i> variations in B6 and CBy mice.

<p>A) The autoradiographs show representative SP-PCR analyses of DNA, extracted from heart, liver, striatum and tail. At weaning the B6.Cg-R6/1 (B6) and CBy.Cg-R6/1 (CBy) congenic mice contained in tail DNA (CAG)98 and (CAG)94, respectively. For comparison the profiles of the <i>Msh2</i>−/− mouse is shown. About 5–10 DNA amplifiable molecules were amplified in each reaction with primers MS-1F and MS-1R. Animals were 20-weeks old. B) Congenic CBy.Cg-R6/1 mice were crossed to B6 and the resulting F1 progeny were crossed to produce F2 mice with all possible genotypes at the <i>Msh3</i> locus. Repeat instability was assayed by amplifying 10 ng genomic DNA using fluorescently labelled primers and resolving the fragments by capillary gel electrophoresis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen-1003280-g001" target="_blank">Figure 1B</a>). Using this high-resolution approach repeat length distributions present with the typical ‘hedgehog’ pattern (<i>e.g. </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Mangiarini1" target="_blank">[10]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Manley1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Wheeler1" target="_blank">[15]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-vanDenBroek1" target="_blank">[16]</a>. This pattern reflects both somatic mosaicism within the sample and PCR artefacts generated by <i>Taq</i> polymerase slippage <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Zhang1" target="_blank">[62]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003280#pgen.1003280-Hauge1" target="_blank">[63]</a>. The PCR artefacts are predominantly repeat contractions, hence these are not considered here. The pattern of CAG repeat instability depended on genotype at the MSH3 locus. B6 homozygosity resulted in the greatest instability, CBy homozygosity resulted in lack of expansion, while heterozygosity resulted in an intermediate instability, indicative of a gene dosage effect of the <i>Msh3</i> locus. Numbers indicate the CAG repeat size corresponding to major peaks. In addition, on the B6 tracing, a second number indicates the highest CAG repeat number detected. C) <i>Msh3</i> polymorphisms in <i>Msh3</i> gene from C57BL/6 (B6) and BALB/cBy (CBy) mice. Promoters were identical. SNPs were identified or confirmed to those in <i>dbSNP</i> by sequencing the <i>Msh3</i> gene.</p