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Type IA topoisomerase inhibition by clamp closure

Leelaram, Majety Naga and Bhat, Anuradha Gopal and Godbole, Adwait Anand and Bhat, Rajeshwari Subray and Manjunath, Ramanathapuram and Nagaraja, Valakunja (2013) Type IA topoisomerase inhibition by clamp closure. In: FASEB JOURNAL, 27 (8). pp. 3030-3038.

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Official URL: http://dx.doi.org/10.1096/fj.12-226118

Abstract

Bacterial DNA topoisomerase I (topoI) catalyzes relaxation of negatively supercoiled DNA. The enzyme alters DNA topology through protein-operated DNA gate, switching between open and closed conformations during its reaction. We describe the mechanism of inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis topoI by monoclonal antibodies (mAbs) that bind with high affinity and inhibit at 10-50 nM concentration. Unlike other inhibitors of topoisomerases, the mAbs inhibited several steps of relaxation reaction, namely DNA binding, cleavage, strand passage, and enzyme-DNA dissociation. The enhanced religation of the cleaved DNA in presence of the mAb indicated closing of the enzyme DNA gate. The formation of enzyme-DNA heterocatenane in the presence of the mAbs as a result of closing the gate could be inferred by the salt resistance of the complex, visualized by atomic force microscopy and confirmed by fluorescence measurements. Locking the enzyme-DNA complex as a closed clamp restricted the movements of the DNA gate, affecting all of the major steps of the relaxation reaction. Enzyme trapped on DNA in closed clamp conformation formed roadblock for the elongating DNA polymerase. The unusual multistep inhibition of mycobacterial topoisomerases may facilitate lead molecule development, and the mAbs would also serve as valuable tools to probe the enzyme mechanism.

Item Type: Journal Article
Additional Information: Copyright for this article belongs to the FEDERATION AMER SOC EXP BIOL, USA
Keywords: DNA cleavage-religation; enzyme-DNA heterocatenane; monoclonal antibody; mycobacteria
Department/Centre: Division of Biological Sciences > Microbiology & Cell Biology
Depositing User: Id for Latest eprints
Date Deposited: 24 Feb 2014 07:56
Last Modified: 22 Nov 2018 15:04
URI: http://eprints.iisc.ac.in/id/eprint/48448

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