DNA mismatch repair during replication is a conserved process essential for maintaining genomic stability. Mismatch repair is also implicated in cell-cycle arrest and apoptosis after DNA damage. Because yeast and human mismatch repair systems are well conserved, we have employed the budding yeast Saccharomyces cerevisiae to understand the regulation and function of the mismatch repair gene MSH2. Using a luciferase-based transcriptional reporter, we defined a 218-bp region upstream of MSH2 that contains cell-cycle and DNA damage responsive elements. The 5' end of the MSH2 transcript was mapped by primer extension and was found to encode a small upstream open reading frame (uORF). Mutagenesis of the uORF start codon or of the uORF stop codon, which creates a continuous reading frame with MSH2, increased Msh2 steady-state protein levels ~2-fold. Furthermore, we found that the cell-cycle transcription factors Swi6, Swi4, and Mbp1-along with SCB/MCB cell-cycle binding sites upstream of MSH2-are all required for full basal expression of MSH2. Mutagenesis of the cell-cycle boxes resulted in a minor reduction in basal Msh2 levels and a 3-fold defect in mismatch repair. Disruption of the cell-cycle boxes also affected growth in a DNA polymerase-defective strain background where mismatch repair is essential, particularly in the presence of the DNA damaging agent methyl methane sulfonate (MMS). Promoter replacements conferring constitutive expression of MSH2 revealed that the transcriptional induction in response to MMS is required to maintain induced levels of Msh2. Turnover experiments confirmed an elevated rate of degradation in the presence of MMS. Taken together, the data show that the DNA damage regulation of Msh2 occurs at the transcriptional and post-transcriptional levels. The transcriptional and translational control elements identified are conserved in mammalian cells, underscoring the use of yeast as a model system to examine the regulation of MSH2.
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