Defects in DNA repair pathways are associated with genome instability and carcinogenesis. DNA deamination results in conversion of adenosine to inosine, cytosine to uridine, and guanosine to xanthosine. Endonuclease V mediates an evolutionary conserved pathway involved in inosine repair. The long-term objective of this proiect is to elucidate the molecular mechanism underlying the endonuclease V-mediated repair pathway and understand its cellular functions in humans. The short-term goal of this proposal is to establish a cell-free repair system in Escherichia coli and explore its use in identifying cellular components involved in the endonuclease V-mediated DNA repair pathway. Substrates that can be used in the cell-free assays will be constructed. Protocols for the preparation of E. coli cell-free extracts will be developed. An in vitro assay system will be established to monitor endonuclease V-mediated repair activity. The repair patch created during the repair process will be determined. Biochemical approaches will be employed to identify downstream proteins in fractionated E. coli extracts that interact or displace endonuclease V. Assays will be devised to determine the biochemical functions of the identified proteins. The cell-free assay will be used to validate the roles of the downstream proteins in inosine repair, and identify additional proteins such as DNA polymerase required in this pathway. Insights gained from this study will provide a biochemical framework for investigation of human endonuclease V-mediated repair pathway.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM067744-01
Application #
6597146
Study Section
Special Emphasis Panel (ZRG1-F09 (20))
Program Officer
Wolfe, Paul B
Project Start
2003-05-01
Project End
2006-04-30
Budget Start
2003-05-01
Budget End
2006-04-30
Support Year
1
Fiscal Year
2003
Total Cost
$140,000
Indirect Cost
Name
Clemson University
Department
Genetics
Type
Schools of Earth Sciences/Natur
DUNS #
042629816
City
Clemson
State
SC
Country
United States
Zip Code
29634
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Gao, Honghai; Huang, Jianmin; Barany, Francis et al. (2007) Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations. Nucleic Acids Res 35:e2
Lin, Jun; Gao, Honghai; Schallhorn, Kathryn A et al. (2007) Lesion recognition and cleavage by endonuclease V: a single-molecule study. Biochemistry 46:7132-7
Zhang, Zhongge; Ma, Che; Pornillos, Owen et al. (2007) Functional characterization of the heterooligomeric EbrAB multidrug efflux transporter of Bacillus subtilis. Biochemistry 46:5218-25
Feng, Hong; Dong, Liang; Cao, Weiguo (2006) Catalytic mechanism of endonuclease v: a catalytic and regulatory two-metal model. Biochemistry 45:10251-9
Feng, Hong; Dong, Liang; Klutz, Athena M et al. (2005) Defining amino acid residues involved in DNA-protein interactions and revelation of 3'-exonuclease activity in endonuclease V. Biochemistry 44:11486-95
Feng, Hong; Klutz, Athena M; Cao, Weiguo (2005) Active site plasticity of endonuclease V from Salmonella typhimurium. Biochemistry 44:675-83
Hitchcock, Thomas M; Gao, Honghai; Cao, Weiguo (2004) Cleavage of deoxyoxanosine-containing oligodeoxyribonucleotides by bacterial endonuclease V. Nucleic Acids Res 32:4071-80
Cao, Weiguo (2004) Recent developments in ligase-mediated amplification and detection. Trends Biotechnol 22:38-44
Lu, Jing; Tong, Jie; Feng, Hong et al. (2004) Unique ligation properties of eukaryotic NAD+-dependent DNA ligase from Melanoplus sanguinipes entomopoxvirus. Biochim Biophys Acta 1701:37-48

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