The DNA mismatch repair (MMR) system corrects DNA synthesis errors that occur during replication and also is involved in several other DNA transactions. MMR is initiated by MutS and MutL homologs, which are highly conserved throughout prokaryotes and eukaryotes. They are both dimers and contain DNA binding and ATPase activities that are essential for MMR in vivo. Inactivation of these proteins leads to increased mutagenesis, improper recombination, and resistance to the cytotoxic effects of several DNA damaging agents. In humans, mutations in the mismatch repair genes are directly linked to hereditary non-polyposis colorectal cancer (HNPCC) and are associated with several sporadic cancers. Because of the diversity of functions carried out by the MMR proteins, it will be essential to understand the molecular mechanisms that underlie these different processes to develop effective treatment for the associated diseases and cancers. In eukaryotes, MutS? (MSH2-MSH6) and MutL? (MLH1-PMS2) are the primary MutS and MutL homologs responsible for initiation of MMR. MutS? initiates repair by binding to a mismatch and undergoing an ATP-dependent conformational change that promotes its interaction with MutL?. PCNA then activates MutL? to incise the daughter strand both 5'and 3' to the mismatch. Subsequently, MutS? activates the 5'-3'exonuclease EXO1 to processively excise the DNA containing the incorrect nucleotide. Finally, DNA polymerase ? or ? catalyzes resynthesis, and DNA ligase seals the nick. Structural and biochemical studies, including several from our lab, indicate that the conformational dynamics and assembly states of the proteins and protein-DNA complexes are central to the regulation of MMR. The overall goal of this proposal is to elucidate the structure-function relationships that govern the initiation steps of MMR. We propose a systematic series of experiments, in which we characterize the structural, conformational, and dynamic properties of MMR complexes formed with MutS? using atomic force microscopy (AFM), our newly developed Dual Resonance frequency Enhanced Electrostatic force Microscopy (DREEM), which allows visualization of the path of the DNA through the proteins, and single-molecule fluorescence. These studies will be complemented with a thorough examination of the biochemical and functional properties done by our collaborators in Paul Modrich's and Peggy Hsieh's laboratories. This combination of techniques will allow us to fully characterize the binding, dynamic, conformational, and functional properties of complexes that govern MMR and the preservation of genomic stability. Our goals are to: 1) dissect the molecular mechanisms of mismatch recognition by MutS?, and 2) determine the conformational properties of MutS?-MutL?-mismatch complexes that govern the initiation of repair.

Public Health Relevance

DNA mismatch repair proteins correct errors generated during DNA replication and also are involved in several other processes, including DNA-damage-induced apoptosis, recombination, double-strand break repair. Inactivation of mismatch repair proteins dramatically increases the frequency of mutations, decreases apoptosis, increases cell survival, causes resistance to chemotherapy, and is directly linked to hereditary non-polyposis colorectal cancer and associated with several sporadic cancers in humans. The central goal of this grant is to elucidate the basic mechanisms that underlie the initiation of mismatch repair, which will be especially important for designing treatment of cancers in which mismatch repair is compromised.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM079480-05A1
Application #
8630482
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Janes, Daniel E
Project Start
2007-08-01
Project End
2018-02-28
Budget Start
2014-04-10
Budget End
2015-02-28
Support Year
5
Fiscal Year
2014
Total Cost
$301,575
Indirect Cost
$84,956
Name
University of North Carolina Chapel Hill
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Erie, Dorothy A; Weninger, Keith R (2014) Single molecule studies of DNA mismatch repair. DNA Repair (Amst) 20:71-81
DeRocco, Vanessa C; Sass, Lauryn E; Qiu, Ruoyi et al. (2014) Dynamics of MutS-mismatched DNA complexes are predictive of their repair phenotypes. Biochemistry 53:2043-52
Archer, Eva J; Simpson, Mark A; Watts, Nicholas J et al. (2013) Long-range architecture in a viral RNA genome. Biochemistry 52:3182-90
Geng, Hui; Sakato, Miho; DeRocco, Vanessa et al. (2012) Biochemical analysis of the human mismatch repair proteins hMutS? MSH2(G674A)-MSH6 and MSH2-MSH6(T1219D). J Biol Chem 287:9777-91
Qiu, Ruoyi; DeRocco, Vanessa C; Harris, Credle et al. (2012) Large conformational changes in MutS during DNA scanning, mismatch recognition and repair signalling. EMBO J 31:2528-40
Kennedy, Scott R; Erie, Dorothy A (2011) Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription. Proc Natl Acad Sci U S A 108:6079-84
DeRocco, Vanessa; Anderson, Trevor; Piehler, Jacob et al. (2010) Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes. Biotechniques 49:807-16
Sass, Lauryn E; Lanyi, Cherie; Weninger, Keith et al. (2010) Single-molecule FRET TACKLE reveals highly dynamic mismatched DNA-MutS complexes. Biochemistry 49:3174-90
Chelico, Linda; Prochnow, Courtney; Erie, Dorothy A et al. (2010) Structural model for deoxycytidine deamination mechanisms of the HIV-1 inactivation enzyme APOBEC3G. J Biol Chem 285:16195-205
Erie, Dorothy A; Kennedy, Scott R (2009) Forks, pincers, and triggers: the tools for nucleotide incorporation and translocation in multi-subunit RNA polymerases. Curr Opin Struct Biol 19:708-14

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