Abstract
The DNA mismatch repair (MMR) pathway corrects specific types of DNA replication errors that affect microsatellites and thus is critical for maintaining genomic integrity. The genes of the MMR pathway are highly conserved across different organisms. Likewise, defective MMR function universally results in microsatellite instability (MSI) which is a hallmark of certain types of cancer associated with the Mendelian disorder hereditary nonpolyposis colorectal cancer. (Lynch syndrome). To identify previously unrecognized deleted genes or loci that can lead to MSI, we developed a functional genomics screen utilizing a plasmid containing a microsatellite sequence that is a host spot for MSI mutations and the comprehensive homozygous diploid deletion mutant resource for Saccharomyces cerevisiae. This pool represents a collection of non-essential homozygous yeast diploid (2N) mutants in which there are deletions for over four thousand yeast open reading frames (ORFs). From our screen, we identified a deletion mutant strain of the PAU24 gene that leads to MSI. In a series of validation experiments, we determined that this PAU24 mutant strain had an increased MSI-specific mutation rate in comparison to the original background wildtype strain, other deletion mutants and comparable to a MMR mutant involving the MLH1 gene. Likewise, in yeast strains with a deletion of PAU24, we identified specific de novo indel mutations that occurred within the targeted microsatellite used for this screen.
Highlights
The DNA mismatch repair (MMR) pathway corrects specific types of replication errors caused by DNA polymerase slippage and is critical for maintaining genomic integrity
We previously demonstrated that this chimeric Ura3p protein permits growth on media lacking uracil (Ura-) and failure to grow in 5-fluorotic acid (FOA) containing media [5]
Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC) is caused by germline mutations in one of several DNA mismatch repair (MMR) genes, namely MLH1, MSH2, MSH6 and PMS2 [1]
Summary
The DNA mismatch repair (MMR) pathway corrects specific types of replication errors caused by DNA polymerase slippage and is critical for maintaining genomic integrity. The canonical genes of the MMR pathway are highly conserved among different species including Escherichia coli, Saccharomyces cerevisiae and Homo sapiens. Defective MMR function increases the rate of insertion and deletion (indels) mutations in microsatellites and this molecular phenotype is commonly referred as microsatellite instability (MSI). In H. sapiens, the consequences of defective DNA MMR is dramatically apparent in the Mendelian cancer syndrome, hereditary nonpolyposis colorectal cancer (HNPCC), otherwise known as Lynch Syndrome [1]. Affected individuals have germline mutations in the human MMR genes MSH2, MLH1, PMS2 and MSH6, and are at substantially increased risk for developing MSI-positive colorectal carcinoma as well as other malignancies including endometrial, gastrointestinal and genitourinary cancers [2]. Not all clinical cases of Lynch syndrome have been explained by germline mutations in one of these four genes, suggesting that other genes may contribute
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