The long-term goal of this project is to understand the molecular mechanism of human mismatch repair (MMR) and its impact on human health and disease. The importance of MMR is underscored by the fact that defects in MMR genes lead to severe genomic instability and eventually to cancer predisposition, including hereditary non-polyposis colorectal cancer (HNPCC) and certain types of sporadic cancers. MMR in human cells is nick-directed and involves at least nine proteins, which are MutS alpha, MutS beta, MutL alpha, EXO1, polymerase delta, PCNA, RFC, RPA, and HMGB1. Despite extensive investigations on the initiation of mismatch-provoked excision, which appears to require MutS alpha, PCNA, RPA, and EXO1. However, how mismatch recognition by MutS proteins leads to a strand-specific excision at a single-stranded break several hundred base pairs away from the mismatch is not understood. Three current models on mismatch- provoked excision are quite controversial in terms of how many MutS molecules are required and how the strand discrimination signal is recognized. Recently, mismatch-provoked excision has been reconstituted. Interestingly, EXO1, a 5'?>3' exonuclease, was reported to be capable of bi-directional removal of mismatch; and EXO1-catalyzed 5' excision is independent of PCNA. Given the importance of PCNA in mismatch- provoked excision and the involvement of four exonucleases in the E. coli methyl-direct MMR, we hypothesize that multiple nucleases are involved in the human reaction. Indeed, we have recently identified a novel 5'?>3' mismatch excision pathway, which depends on at least MutS alpha, PCNA, a novel 5' nuclease, and a stimulating factor. This application proposes to purify and characterize both the novel 5' nuclease and the excision stimulating factor, and eventually reconstitute the PCNA-dependent 5' excision reaction in vitro using purified proteins. The purified system will then be used to evaluate three controversial models to elucidate the molecular mechanism of mismatch-provoked excision. Since defects in mismatch excision genes, e.g., the EXO1 gene, are associated with cancer development, identifying the components required for the novel 5' excision pathway will provide new diagnostic markers for HNPCC and other MMR deficient cancer syndromes. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA115942-03
Application #
7407362
Study Section
Special Emphasis Panel (ZRG1-GGG-E (90))
Program Officer
Okano, Paul
Project Start
2006-07-01
Project End
2011-05-31
Budget Start
2008-06-01
Budget End
2009-05-31
Support Year
3
Fiscal Year
2008
Total Cost
$227,246
Indirect Cost
Name
University of Kentucky
Department
Pharmacology
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
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Mao, Guogen; Lee, Sanghee; Ortega, Janice et al. (2012) Modulation of microRNA processing by mismatch repair protein MutL?. Cell Res 22:973-85
Tian, Lei; Gu, Liya; Li, Guo-Min (2009) Distinct nucleotide binding/hydrolysis properties and molar ratio of MutSalpha and MutSbeta determine their differential mismatch binding activities. J Biol Chem 284:11557-62
Hou, Caixia; Chan, Nelson L S; Gu, Liya et al. (2009) Incision-dependent and error-free repair of (CAG)(n)/(CTG)(n) hairpins in human cell extracts. Nat Struct Mol Biol 16:869-75
Tian, Lei; Hou, Caixia; Tian, Keli et al. (2009) Mismatch recognition protein MutSbeta does not hijack (CAG)n hairpin repair in vitro. J Biol Chem 284:20452-6
Zhang, Yanbin; Yuan, Fenghua; Wang, Daojing et al. (2008) Identification of regulatory factor X as a novel mismatch repair stimulatory factor. J Biol Chem 283:12730-5
Li, Guo-Min (2008) Mechanisms and functions of DNA mismatch repair. Cell Res 18:85-98