The long-term objectives of this project are to understand the molecular mechanism of human mismatch repair and its impact on human cancer. DNA mismatch repair plays a crucial role in maintaining genomic stability by correcting mismatches generated from DNA biosynthetic errors and DNA recombination. Defects in human mismatch repair are the primary cause of both hereditary colorectal cancer and sporadic colorectal cancers that display microsatellite instability. Microsatellite instability, which correlates with mismatch repair deficiency, has also been demonstrated in a substantial fraction of many types of sporadic cancer, including bladder cancers. Recently, sporadic bladder cancers have been shown to display a higher rate of microsatellite instability than other sporadic cancers. The goals of this application are to determine if mismatch repair deficiency is associated with sporadic bladder cancers and to isolate and characterize novel mismatch repair components/genes. Experiments will be developed in the following three specific areas. 1) The mismatch repair proficiency of bladder cell lines with microsatellite instability will be determined using an in vitro biochemical mismatch repair assay. 2) Novel mismatch repair activities will be first characterized by complementation experiments using the known mismatch repair proteins and/or mutant cell lines, and then be purified from HeLa nuclear extracts by virtue of their ability to restore mismatch repair to the novel mutant cell lines. 3) The gene(s) encoding the novel protein(s) will be cloned by the """"""""reverse genetic"""""""" approach. Peptide sequences will be obtained from the novel protein(s) and used for designing degenerate primers to amplify DNA fragments of interest, which are in turn used to identify full length cDNAs. This study will not only provide insight into the etiology of sporadic bladder cancers, but also lead to the identification of novel mismatch repair components.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA085377-04
Application #
6697062
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Okano, Paul
Project Start
2001-02-14
Project End
2006-01-31
Budget Start
2004-02-01
Budget End
2006-01-31
Support Year
4
Fiscal Year
2004
Total Cost
$152,945
Indirect Cost
Name
University of Kentucky
Department
Pharmacology
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
Murata, Hiroaki; Khattar, Nada H; Gu, Liya et al. (2005) Roles of mismatch repair proteins hMSH2 and hMLH1 in the development of sporadic breast cancer. Cancer Lett 223:143-50
Zhang, Yanbin; Yuan, Fenghua; Presnell, Steven R et al. (2005) Reconstitution of 5'-directed human mismatch repair in a purified system. Cell 122:693-705
Guo, Shuangli; Presnell, Steven R; Yuan, Fenghua et al. (2004) Differential requirement for proliferating cell nuclear antigen in 5' and 3' nick-directed excision in human mismatch repair. J Biol Chem 279:16912-7
Yuan, Fenghua; Gu, Liya; Guo, Shuangli et al. (2004) Evidence for involvement of HMGB1 protein in human DNA mismatch repair. J Biol Chem 279:20935-40
Li, Guo-Min (2003) DNA mismatch repair and cancer. Front Biosci 8:d997-1017
McCulloch, Scott D; Gu, Liya; Li, Guo-Min (2003) Nick-dependent and -independent processing of large DNA loops in human cells. J Biol Chem 278:50803-9
McCulloch, Scott D; Gu, Liya; Li, Guo-Min (2003) Bi-directional processing of DNA loops by mismatch repair-dependent and -independent pathways in human cells. J Biol Chem 278:3891-6
Gu, Liya; Wu, Jianxin; Zhu, Bei-Bei et al. (2002) Deficiency of a novel mismatch repair activity in a bladder tumor cell line. Nucleic Acids Res 30:2758-63
Ramilo, Cecilia; Gu, Liya; Guo, Shuangli et al. (2002) Partial reconstitution of human DNA mismatch repair in vitro: characterization of the role of human replication protein A. Mol Cell Biol 22:2037-46
Gu, Liya; Cline-Brown, Brandee; Zhang, Fujian et al. (2002) Mismatch repair deficiency in hematological malignancies with microsatellite instability. Oncogene 21:5758-64