Mismatch repair (MMR) improves the fidelity of DNA replication by excising mismatches that result from DNA polymerase misincorporation errors. Msh proteins initiate MMR by binding to DNA mismatches and then interact with Mlh proteins to recruit downstream repair factors that excise the newly replicated strand containing the mismatch. Mutations in MSH and MLH genes confer significant increases (~1000X) in mutation rate and have been implicated in hereditary forms of colon cancer (HNPCC). The mechanisms by which MMR proteins, and in particular Mlh proteins, signal downstream repair and recombination factors are not well understood. We are focused on understanding how two Mlh complexes, Mlh1-Pms1 and Mlh1-Mlh3, function in DNA repair signaling and how genetic incompatibilities between Mlh factors can lead to increased mutation rates. The latter work can provide a better understanding of how cells evolve to become resistant to growth control and therapeutics.
In Aim 1 we are analyzing the behavior of single Mlh complexes interacting with DNA using total internal fluorescence microscopy. We are interested in answering two critical questions: 1. How do the Mlh and Msh proteins interact with PCNA on a mismatch DNA template? 2. How does Mlh1-Pms1 bypass obstacles while identifying downstream targets? These studies will take advantage of mutants generated in the lab and are aimed at developing accurate models for early steps in MMR that cannot be accomplished using bulk approaches.
Aim 2 is focused on characterizing a role for the Mlh1-Mlh3 complex in MMR and meiotic recombination. We propose to examine the functions of Mlh1-Mlh3 in genetic and biochemical assays, with the goal of understanding how this complex acts in two seemingly unrelated processes. Specifically, will perform an alanine-scan mutagenesis of MLH3 and test the effect of these mutations in MMR and meiotic crossover assays, and willl purify wild-type and mutant Mlh1-Mlh3 to examine interactions with Msh-mismatch complexes in bulk and single-molecule assays, and also test whether the putative endonuclease domain of Mlh1-Mlh3 acts on a variety of substrates including those containing mismatch loops and structures predicted to be recombination intermediates.
Aim 3 uses MMR incompatibilities as a model to study adaptive evolution and disease progression. MLH incompatibility leads to an elevated mutation rate, which has the potential to increase the rate of both adaptive and deleterious mutations. We are interested in testing if a MMR incompatibility will initially increase evolvability through acquirig mutations, both beneficial and deleterious, allowing fixation of adaptive mutations in large populations through reacquisition of MMR functions by subsequent mating/recombination. We will assess the fitness of compatible and incompatible MLH combinations in non-selective and selective conditions. Such experiments, combined with methods used previously to identify mutations responsible for DNA damage sensitivity and recessive lethality phenotypes, can also provide insights into understanding how driver mutations arise in HNPCC and other cancers.
Colon cancer is the fourth most common cancer to affect Americans (141,000 new cases estimated in 2011);mutations in DNA mismatch repair genes are linked to a hereditary form of colorectal cancer, HNPCC, which represents ~2-7% of colon cancers. The high conservation of mismatch repair factors between yeast and humans makes yeast an ideal model system to study this repair process using genetic, biochemical and whole genome sequencing approaches. HNPCC has a high cure rate if detected early, underscoring the importance of obtaining new mechanistic understandings of mismatch repair and new diagnostic tools.
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