SEQUENCE-SPECIFIC INTERACTION OF MYCOBACTERIUM-SMEGMATIS TOPOISOMERASE-I WITH DUPLEX DNA

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electro phoretic mobility shift assay of enzyme-DNA covalent complexes; Mg2+, an essential component for DNA relaxation activity of the enzyme, is n ot required for binding to DNA, The enzyme makes single-stranded nicks , with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More impo rtantly, the enzyme binds to duplex DNA having a preferred site with h igh affinity, a. property similar to the eukaryotic type I topoisomera ses, The preferred cleavage site is mapped on a 65 bp duplex DNA and f ound to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eu karyotic enzymes in DNA recognition properties.