DNA topoisomerases catalyze DNA topological changes and affect vital cellular processes including replication, transcription, recombination and DNA repair. They are targets of a number of anti-bacterial and anti-cancer drugs. Presently, there is no specific inhibitor directed toward E. coli DNA topoisomerase I. Because there is little homology between the bacterial and mammalian type I DNA topoisomerases, a specific inhibitor against the bacterial DNA topoisomerase I should be a useful antibiotic. The details of the mechanism and structure of topoisomerases remain to be elucidated. This proposal is aimed towards that goal. There are three zinc atoms in each molecule of E. coli DNA topoisomerase I. The amino acid sequence of the likely zinc binding motifs CX2CGX2MX12- 13CX4-10C with four cysteines as coordination site is significantly different from the zinc fingers in eukaryotic DNA-binding proteins where zinc is coordinated to two cysteines and two histidines, suggesting that the structure of these zinc binding domains is likely to be different. This proposal seeks to characterize the contributions of the zinc atoms in DNA topoisomerase enzyme structure and function. It remains to be determined if the zinc atoms are required for proper folding, or involved directly in DNA binding and catalysis of topoisomerization. Limited proteolysis, DNA binding assays, ultra-violet and fluorescence spectroscopy will be used to determine the differences between apo- and metal containing topoisomerase I. Oligonucleotide-directed mutants in the proposed zinc binding motifs and random mutants that require zinc for topoisomerase in vivo function will be constructed. These genetic studies will complement ongoing effort of determination of the 3-dimensional structure of the enzyme. Because a zinc binding motif with the same consensus sequence is also present in the E. coli uvrA protein, the knowledge gained from this work may also apply to other DNA binding proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042774-04
Application #
3301633
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1990-04-01
Project End
1995-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
New York Medical College
Department
Type
Schools of Medicine
DUNS #
City
Valhalla
State
NY
Country
United States
Zip Code
10595