TY - JOUR
T1 - Characterization of DNA topoisomerase i from Mycobacterium tuberculosis
T2 - DNA cleavage and religation properties and inhibition of its activity
AU - Godbole, Adwait Anand
AU - Leelaram, Majety Naga
AU - Bhat, Anuradha Gopal
AU - Jain, Paras
AU - Nagaraja, Valakunja
N1 - Funding Information:
The authors acknowledge the members of the laboratory for critical reading of the manuscript and useful suggestions. B. Mallick and S.M. Hegde are acknowledged for technical assistance. V.N. is a J.C. Bose fellow of Department of Science and Technology, and a recipient of the Centre for Excellence grant from Department of Biotechnology, Government of India and partner 14 in an EU consortium project, MM4TB.
PY - 2012/12/15
Y1 - 2012/12/15
N2 - Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg2+ 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 Mg2+ 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.
AB - Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg2+ 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 Mg2+ 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.
KW - DNA cleavage
KW - DNA relaxation
KW - Mycobacterium tuberculosis
KW - Topoisomerase I
KW - Toprim motif
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U2 - 10.1016/j.abb.2012.10.004
DO - 10.1016/j.abb.2012.10.004
M3 - Article
C2 - 23085346
AN - SCOPUS:84868453744
SN - 0003-9861
VL - 528
SP - 197
EP - 203
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 2
ER -