Mismatch repair (MMR) improves the fidelity of DNA replication by about 1000 fold by excising mismatches in the newly replicated strand arising from mis-incorporation and DNA slippage. MSH proteins initiate MMR by binding to DNA mismatches and then interacting with MLH proteins to recruit downstream repair factors. Mutations in MSH and MLH genes confer significant increases in mutation rate. MMR factors also prevent recombination between divergent DNA sequences, and process recombination intermediates containing nonhomologous ? single-stranded ends. The mechanisms by which these proteins identify mismatches and signal ? downstream factors during DNA replication and recombination are not well understood. In addition, the role of genetic background in determining the penetrance of MMR mutations with respect to disease phenotype has not been explored in depth. This proposal is focused on understanding how MMR proteins identify mismatches and signal downstream factors during DNA replication and repair, and the role of genetic background in determining the penetrance of MMR mutations.
In Aim 1 we will analyze the behavior of single MSH and MLH complexes interacting with DNA using total internal fluorescence microscopy. These studies will take advantage of a large number of MMR mutants generated previously in the lab and are aimed at distinguishing between competing models for how MSH and MLH proteins signal downstream steps in MMR.
In Aim 2 we will use SNP scanner technology to examine genome-wide mutation accumulation in MMR mutants. This work will allow us to determine the actual mutation rate in a MMR-defective strain and provide information that should help cancer researchers distinguish mutations critical for transformation to a cancer state from those that occur after transformation.
Aim 3 is also focused on genome stability and presents in vitro and in vivo ? biochemical approaches to test interactions between MMR components and the SGS1 helicase to prevent recombination between divergent DNA sequences.
Aim 4 outlines experiments aimed at understanding how genome instabilities arise from genetic incompatibility in MMR. This work will provide models to explain how genetic background contributes to disease penetrance in humans. In addition it offers new tools to identify genetic interactions in DNA repair pathways, with the overall goal of understanding cancer susceptibility and the molecular pathways that function in DNA repair. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM053085-14
Application #
7462696
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Portnoy, Matthew
Project Start
1995-08-01
Project End
2012-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
14
Fiscal Year
2008
Total Cost
$373,040
Indirect Cost
Name
Cornell University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
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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