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Abstract
Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.
Highlights
DNA mismatch repair (MMR) acts to repair the potentially mutagenic misincorporation errors that occur during normal DNA replication and the absence of MMR results in increased rates of accumulating mutations
Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/ scPMS1
Microscopy and protein biochemistry to analyze the model organism Saccharomyces cerevisiae to further characterize a poorly understood mismatch repair pathway that functions in the absence of EXO1 that is highly dependent on the Mlh1-Pms1 complex
Summary
DNA mismatch repair (MMR) acts to repair the potentially mutagenic misincorporation errors that occur during normal DNA replication and the absence of MMR results in increased rates of accumulating mutations. MMR repairs mispaired bases that occur in recombination intermediates as well as prevents inappropriate recombination between DNAs with imperfect homology where recombination could result in genome rearrangements [8,9,10]. In E. coli MMR, mismatches are recognized by the MutS homodimer [15,16]. Mispair bound MutS recruits the MutL homodimer [17]. This recruitment leads to activation of the MutH endonuclease, which introduces single strand breaks, called nicks, at unmethylated GATC sites in the newly replicated and hemimethylated DNA strand [18]. A combination of the UvrD helicase and one of four single stranded DNA specific exonucleases excise the nicked strand past the mispair and the resulting singled-stranded gap is filled in by DNA polymerase III, single strand DNA binding protein and DNA ligase [14,19]
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