Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity.

Adwait Anand Godbole, Majety Naga Leelaram, Anuradha Gopal Bhat, Paras Jain, Valakunja Nagaraja

Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.

Archives of biochemistry and biophysics 2012 Dec 15

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Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg(2+) dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg(2+) co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg(2+) for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors. Copyright © 2012 Elsevier Inc. All rights reserved.

Citation

Adwait Anand Godbole, Majety Naga Leelaram, Anuradha Gopal Bhat, Paras Jain, Valakunja Nagaraja. Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity. Archives of biochemistry and biophysics. 2012 Dec 15;528(2):197-203