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Escherichia coli topoisomerase IV. Purification, characterization, subunit structure, and subunit interactions
1Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York.
The Journal of Biological Chemistry
|November 15, 1993
Summary
Researchers identified full-length ParC and ParE proteins as essential for reconstituting active topoisomerase IV (Topo IV) enzyme activity in E. coli. Truncated versions were inactive, highlighting the importance of specific protein domains for enzyme function.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Topoisomerase IV (Topo IV) is a crucial enzyme in DNA replication and segregation.
- Understanding the structure-function relationship of Topo IV subunits (ParC and ParE) is vital for bacterial genetics.
- Previous studies had reported different lengths for E. coli ParC and ParE subunits.
Purpose of the Study:
- To analyze the DNA sequences of Escherichia coli parC and parE genes.
- To investigate the role of full-length versus truncated ParC and ParE proteins in Topo IV activity.
- To characterize the biochemical properties and quaternary structure of active Topo IV.
Main Methods:
- DNA sequence analysis of E. coli parC and parE genes.
- Overproduction of ParC and ParE proteins using T7 RNA polymerase-based expression plasmids.
- Reconstitution of Topo IV activity with full-length and truncated subunits.
- Enzyme kinetics assays using ATP or dATP.
- Antibiotic inhibition studies with quinolones and coumarins.
- Analysis of DNA cleavage patterns induced by norfloxacin.
- Determination of native and active quaternary structures.
Main Results:
- Full-length ParC and ParE subunits were required for reconstituting Topo IV activity; truncated versions were inactive.
- Topo IV activity was dependent on ATP or dATP, with specific Michaelis constants for DNA relaxation and decatenation.
- Topo IV activity was inhibited by quinolone and coumarin antibiotics, but at higher concentrations than DNA gyrase.
- Norfloxacin-induced DNA cleavage patterns for Topo IV and DNA gyrase showed distinct yet overlapping profiles.
- The active form of Topo IV was identified as a heterotetramer (ParC2ParE2).
- Inactivity of truncated subunits was linked to their inability to form the heterotetrameric complex.
Conclusions:
- The N-terminal extension of ParC and C-terminal extension of ParE are critical for Topo IV assembly and function.
- Active Topo IV is a ParC2ParE2 heterotetramer, requiring full-length subunits.
- Topo IV exhibits distinct substrate specificities and antibiotic inhibition profiles compared to DNA gyrase.
- The study provides insights into the structural requirements for bacterial topoisomerase activity and potential targets for antibiotics.