This project aims to understand the actions of eukaryotic mismatch repair (MMR) proteins in correcting DNA replication errors and in processing recombination events. In organisms ranging from E. coli to humans, MMR plays an important role in correcting misincorporation errors and slippage events that occur during DNA replication. In E. coli, MutS and MutL play critical roles in MMR; MutS binds to mispairs and MutL interacts with MutS-mispair complexes. Homologs of MutS (Msh) and MutL (Mlh) have been identified in yeast and higher eukaryotes. Studies in bacteria, yeast and humans have also shown that MMR plays an important role in maintaining chromosome stability by preventing recombination between homologous DNA sequences. The molecular mechanisms by which MMR proteins recognize mismatch substrates and transduce this information to downstream repair factors during DNA replication and genetic recombination are not well understood in eukaryotes. These issues will be addressed in the yeast S. cerevisiae through the following approaches: 1. A goal of the 1994 submission was to identify conditional msh2 mutations as they can reveal intermediate steps in a pathway and provide critical reagents for a second site suppressor screen. Genetic suppression analysis will be performed with six well characterized msh2 and four mlh1 conditional MMR mutations to identify interactions between MSH2 and MLH1 and new downstream MMR factors. 2. Biochemical approaches will be used to examine interactions between the Msh2p-Msh6p (mutS homolog) mismatch recognition complex and other DNA repair and replication factors. This analysis will take advantage of a large collection of dominant negative, conditional, and site specific msh2 and msh6 mutations that we isolated. 3. mlh1 mutants show distinct defects in MMR and in crossing over. To test the idea that MLH1 plays unique and distinct roles in each of these processes, we will mutagenize MLH1 with the goal of identifying mlh1 mutations that affect one but not both processes. 4. To understand how MMR factors are recruited to recombination intermediates, we are investigating the association of Msh2p with DNA using ChIP (chromatin crosslinking, immunoprecipitation and PCR). This procedure will be performed in wild type and recombination defective mutants using both plasmid and chromosomal recombination assays.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Genetics Study Section (GEN)
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Wolfe, Paul B
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Cornell University
Schools of Earth Sciences/Natur
United States
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Raghavan, Vandana; Bui, Duyen T; Al-Sweel, Najla et al. (2018) Incompatibilities in Mismatch Repair Genes MLH1-PMS1 Contribute to a Wide Range of Mutation Rates in Human Isolates of Baker's Yeast. Genetics 210:1253-1266
Chakraborty, Ujani; Dinh, Timothy A; Alani, Eric (2018) Genomic Instability Promoted by Overexpression of Mismatch Repair Factors in Yeast: A Model for Understanding Cancer Progression. Genetics 209:439-456
Al-Sweel, Najla; Raghavan, Vandana; Dutta, Abhishek et al. (2017) mlh3 mutations in baker's yeast alter meiotic recombination outcomes by increasing noncrossover events genome-wide. PLoS Genet 13:e1006974
Bui, Duyen T; Friedrich, Anne; Al-Sweel, Najla et al. (2017) Mismatch Repair Incompatibilities in Diverse Yeast Populations. Genetics 205:1459-1471
Manhart, Carol M; Ni, Xiaodan; White, Martin A et al. (2017) The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans. PLoS Biol 15:e2001164
Manhart, Carol M; Alani, Eric (2017) DNA replication and mismatch repair safeguard against metabolic imbalances. Proc Natl Acad Sci U S A 114:5561-5563
Manhart, Carol M; Alani, Eric (2016) Roles for mismatch repair family proteins in promoting meiotic crossing over. DNA Repair (Amst) 38:84-93
Chakraborty, Ujani; Alani, Eric (2016) Understanding how mismatch repair proteins participate in the repair/anti-recombination decision. FEMS Yeast Res 16:
Chakraborty, Ujani; George, Carolyn M; Lyndaker, Amy M et al. (2016) A Delicate Balance Between Repair and Replication Factors Regulates Recombination Between Divergent DNA Sequences in Saccharomyces cerevisiae. Genetics 202:525-40
Gallardo, Ignacio F; Pasupathy, Praveenkumar; Brown, Maxwell et al. (2015) High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging. Langmuir 31:10310-7

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