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 electrophoretic mobility shift assay of enzyme-DNA covalent complexes. Mg2+, an essential component for DNA relaxation activity of the enzyme, is not 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 importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a property similar to the eukaryotic type I topoisomerases. The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.
Bhaduri
T
T
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Sikder
D
D
Nagaraja
V
V
D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009161:Mycobacterium / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004247:DNA; D004264:DNA Topoisomerases, Type I; D008274:Magnesium