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Indispensable, functionally complementing N and C-terminal domains constitute site-specific topoisomerase I.
Paras Jain1, Valakunja Nagaraja
1Department of Microbiology and Cell Biology, Indian Institute of Science, CV Raman Avenue, Bangalore 560012, India.
Journal of Molecular Biology
|February 24, 2006
Summary
Mycobacterium smegmatis topoisomerase I uses distinct N- and C-terminal domains for DNA binding and relaxation. Reconstituting these domains restores enzyme function, highlighting the C-terminal region's role in DNA passage.
Area of Science:
- Molecular Biology
- Enzymology
- Genetics
Background:
- Mycobacterium smegmatis topoisomerase I is an atypical type IA enzyme.
- It exhibits unique DNA binding and sequence-specific contacts during relaxation.
Purpose of the Study:
- To investigate the roles of the N-terminal and C-terminal domains in M. smegmatis topoisomerase I function.
- To elucidate the mechanism of DNA relaxation and binding.
Main Methods:
- Comparing full-length enzyme with truncated N-terminal and C-terminal polypeptides.
- Assessing DNA binding affinity, sequence specificity, cleavage, and religation activities.
- In vitro reconstitution of enzyme function from separate domains.
Main Results:
- The N-terminal fragment retained site-specific binding, cleavage, and religation.
- The C-terminal fragment showed non-specific DNA binding.
- Neither fragment alone catalyzed DNA relaxation; activity was restored upon mixing.
Conclusions:
- The N-terminal domain is crucial for core catalytic activities.
- The C-terminal domain plays an essential role in facilitating single-strand DNA passage.
- The C-terminal region is vital for the overall DNA relaxation mechanism.