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Principal Investigator: Eric E. Alani
Organization: CORNELL UNIVERSITY
Fiscal Year: 2024
Award: $55,992
Funding agency: National Institute of General Medical Sciences
Project Summary
DNA mismatch repair (MMR) systems act to excise misincorporation errors that occur during DNA replication.
In eukaryotes MSH proteins recognize these errors in the context of base-base and insertion/deletion
mismatches and recruit MLH complexes to form ternary complexes that work with replication factors (RPA,
RFC, PCNA) and Exo1 to excise the newly replicated DNA strand through the mismatch site. This is followed
by DNA re-synthesis steps. MMR factors also recognize mismatches that form during strand invasion steps in
homologous recombination; they recruit a helicase complex that unwinds (rejects) recombination intermediates
and allows a new homology search. In addition, subsets of MMR factors act in meiosis to resolve
recombination intermediates into crossovers (COs). In baker’s yeast the majority of meiotic COs are formed in
an interference-dependent pathway in which double Holliday junctions (dHJs), thought to be stabilized by
Msh4-Msh5, are resolved through the actions of STR helicase/topoisomerase, Exo1 nuclease, and the MutLγ
(Mlh1-Mlh3) endonuclease. Our work is focused on developing molecular models to explain how the different
MSH and MLH factors act in the above pathways. This work will enable us to understand how molecular
defects in these factors underlie human infertility and hereditary forms of colon cancer, and how chromosomal
rearrangements can lead to disease. We will test these ideas through three distinct research themes. In
Project 1 we are studying how conformational changes in MLH proteins, mediated by ATP binding and
hydrolysis, are linked to strand specificity steps in MMR and meiotic recombination. We will use genetic
(mutations in intrinsically disordered domains in Mlh1-Pms1 and Mlh1-Mlh3 and force dimerization of MLH
proteins), biochemical (in vitro reconstitution reactions to determine specific roles for MLH proteins in MMR and
mass-spectrometry) and single-molecule (examine diffusion along DNA and how proteins bypass barriers)
approaches. Project 2 is focused on understanding how MutLγ acts to resolve dHJs in the ZMM pathway. Our
work in the current grant period is consistent with MutLγ endonuclease being activated in MMR and meiotic
crossing over through the formation of a MutLγ filament. We will use this information and biochemical, mass
spectrometry, and genetic methods that take advantage of our identification of mlh3 separation of function
mutants to identify MutLγ interacting factors. Our early work encourages us to initially focus on MutLγ
interactions with the Exo1 nuclease, after which we will test identified factors alone and in combination for their
ability to interact with MutLγ to cleave model HJ and dHJ substrates. Project 3 is aimed at understanding how
the decision is made to repair or reject recombination intermediates. We will analyze how mutations in histone
chaperones and deacetylases, separately and in combination, affect anti-recombination, and will employ an
inducible system to provide a temporal and physical measure of these effects. This work will also encourage us
to pursue a genome-wide screen to identify new factors that regulate the repair/rejection decision.
Terms: <Affect><Alleles><Allelomorphs><Aneuploid><Aneuploidy><Baker's Yeast><Binding><Biochemical><Brewer's Yeast><Bypass><Cancers><Cannot achieve a pregnancy><Chaperone><Chromosomal Rearrangement><Chromosome Segregation><Colon Cancer><Colon Carcinoma><Complex><Crossing Over><Cruciform DNA><DNA><DNA Helicases><DNA Recombination><DNA Replication><DNA Sequence><DNA Synthesis><DNA Unwinding Proteins><DNA biosynthesis><DNA crossover><DNA unwinding enzyme><Deacetylase><Defect><Deoxyribonucleic Acid><Difficulty conceiving><Diffusion><Dimerization><Disease><Disorder><Ensure><Eukaryota><Eukaryote><Failure><Familial Nonpolyposis Colon Cancer><Filament><GeneHomolog><Genetic><Genetic Alteration><Genetic Change><Genetic Crossing Over><Genetic Recombination><Genetic defect><Genome Stability><Genomic Stability><Gestation><Grant><HNPCC><Hereditary><Hereditary Colo-rectal Endometrial Cancer Syndrome><Hereditary Colorectal Endometrial Cancer Syndrome><Hereditary Defective Mismatch Repair Syndrome><Hereditary Non-Polyposis Colon Cancer><Hereditary Nonpolyposis Colo-rectal Cancer><Hereditary Nonpolyposis Colo-rectal Neoplasms><Hereditary Nonpolyposis Colon Cancer><Hereditary Nonpolyposis Colorectal Cancer><Hereditary Nonpolyposis Colorectal Neoplasms><Histones><Holliday Junction DNA><Holliday Junctions><Homolog><Homologous Gene><Homologue><Human><Hydrolysis><In Vitro><Infertility><Inherited><Invaded><LS/HNPCC><Link><Lynch Syndrome><Malignant Neoplasms><Malignant Tumor><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Measures><Mediating><Meiosis><Meiotic Recombination><Methods><Mismatch Repair><Modeling><Modern Man><Molecular><Molecular Chaperones><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Modeling Nucleic Acid Biochemistry><Molecular Modeling Protein/Amino Acid Biochemistry><Molecular Models><Molecular Stereochemistry><Mutation><Pathogenicity><Pathway interactions><Patients><Post-Replication Mismatch Repair><Pregnancy><Protein Dimerization><Protein Family><Proteins><Reaction><Recombination><Research><Role><S cerevisiae><S. cerevisiae><Saccharomyces cerevisiae><Sister Chromatid><Site><Specificity><Syndrome><System><Testing><Topoisomerase><Work><base><bases><cancer in the colon><chromosome division><cohesion><conformation><conformational><conformational state><conformationally><conformations><crossover recombination><diffused><diffuses><diffusing><diffusions><endonuclease><fertility cessation><fertility loss><gene repair><genome mutation><genome wide screen><genomic crossover><helicase><hereditary non-polyposis colo-rectal cancer><hereditary non-polyposis colorectal cancer><homologous recombination><indel><infertile><insertion-deletion><insertion-deletion mutation><insertion/deletion><insertion/deletion mutation><inter-homolog crossover><malignancy><meiotic><meiotic crossover><molecular modeling><mutant><neoplasm/cancer><non-sister chromatid exchange><nonsister chromatid exchange><nuclease><pathway><premature><prematurity><prevent><preventing><reconstitute><reconstitution><recruit><repair><repaired><single molecule><social role><tool>