title: Type IA topoisomerase inhibition by clamp closure.
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.
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
authors.forename: Majety Naga
authors.initials: MN
authors.lastname: Leelaram
authors.forename: Anuradha Gopal
authors.initials: AG
authors.lastname: Bhat
authors.forename: Adwait Anand
authors.initials: AA
authors.lastname: Godbole
authors.forename: Rajeshwari Subray
authors.initials: RS
authors.lastname: Bhat
authors.forename: Ramanathapuram
authors.initials: R
authors.lastname: Manjunath
authors.forename: Valakunja
authors.initials: V
authors.lastname: Nagaraja
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053837:DNA Cleavage / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000201:enzymology; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D059003:Topoisomerase Inhibitors / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D009841:Oligonucleotides; D059003:Topoisomerase Inhibitors; D004247:DNA; D004264:DNA Topoisomerases, Type I
keywords: DNA cleavage-religation; enzyme-DNA heterocatenane; monoclonal antibody; mycobacteria