A Determination of MSH2 and MSH6 Cellular Protein Levels in Saccharomyces Cerevisiae Cells
Genomic mutation rates tend to be very low (~10-10 /generation/base) in a wide variety of organisms and most mutations with a phenotypic effect are deleterious (Drake et al. 1998). This would suggest that increased mutation rates are likely to yield a fitness cost and therefore are likely to face negative selection (Heck et al. 2006). Mismatch repair (MMR) is a DNA repair process that acts to maintain mutation rates at very low levels. In eukaryotes, the Msh2-Msh6 protein complex initiates MMR by binding to base-base and single insertion/deletion DNA mismatches that occur primarily due to DNA polymerase misincorporation errors. MMR proteins also function in genetic recombination and DNA alkylation damage toxicity. Elez, et al. (2007) found that reduced levels of the MMR protein MutL in Escherichia coli affected one type of mitotic recombination but did not affect MMR. To test whether the same could be true for homologous MMR proteins in Saccharomyces cerevisiae, I first measured the levels of MMR proteins in haploid cells grown to mid-log phase. Using a quantitative Western blot analysis, I determined that Msh2p and Msh6p are present at 1600 ± 250 (SEM) and 1300 ± 390 (SEM) per cell, respectively. I then performed a mutational analysis of the MSH2 promoter by mutagenizing its seven putative transcription factor binding sites (MacIsaac et al. 2006). The mutations had little effect on transcription of MSH2 or on cellular Msh2p levels. These data suggest that Msh2p and Msh6p are maintained at similar levels in the cell and that small changes to the MSH2 promoter are insufficient to affect its transcription or cellular Msh2 protein levels.
dissertation or thesis