The goal of this research is to understand how human 06-methylguanine-methyltransferase (06meG-MT) recognizes and repairs the DNA alkylation product 06-methylguanine (06meG) and the similar minor product 04-methylthymine (04meT). The repair of these modified bases by 06meG-MT helps protect cells from the mutagenic consequences of alkylation damage. The protein acts by transferring a methyl group (or other alkyl group) from DNA to itself in a suicide reaction, using a cysteine as the alkyl-acceptor. Our general approach is: (i) Express human 06meG-MT in E. coli and purify the protein. (ii) Identify mutant proteins that are defective in the recognition of methylated DNA or in the methyltransfer reaction. Mutants will be obtained by site-directed mutagenesis of the conserved, polar residues that are most likely to be functionally important. A procedure for selection of non-functional 06meG-MT mutants will also be investigated. (iii) Biochemically characterize wild-type and mutant proteins. This includes analysis of methyltransfer kinetics, measurement of the pKa for protonation of the active site cysteine, and determination of the O6meG-DNA binding properties of methyltransfer-defective mutants. (iv) Co-crystallize a methyltransfer-defective mutant of the protein with methylated DNA as a preliminary step in the determination of its structure. These studies will help in understanding how O6meG-MT reduces the mutagenic and carcinogenic effects of an important class of mutagen.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29CA059492-05
Application #
2390768
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Okano, Paul
Project Start
1993-04-01
Project End
1999-03-31
Budget Start
1997-04-01
Budget End
1999-03-31
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218