A novel bipartite mode of binding of M. smegmatis topoisomerase I to its recognition sequence.
We have investigated interaction of Mycobacterium smegmatis topoisomerase I at its specific recognition sequence. DNase I footprinting demonstrates a large region of protection on both the scissile and non-scissile strands of DNA. Methylation protection and interference analyses reveal base-specific contacts within the recognition sequence. Missing contact analyses reveal additional interactions with the residues in both single and double-stranded DNA, and hence underline the role for the functional groups associated with those bases. These interactions are supplemented by phosphate contacts in the scissile strand. Conformation specific probes reveal protein-induced structural distortion of the DNA helix at the T-A-T-A sequence 11 bp upstream to the recognition sequence. Based on these footprinting analyses that define parameters of topoisomerase I-DNA interactions, a model of topoisomerase I binding to its substrate is presented. Within the large protected region of 30 bp, the enzyme makes direct contact at two locations in the scissile strand, one around the cleavage site and the other 8-12 bases upstream. Thus the enzyme makes asymmetric recognition of DNA and could carry out DNA relaxation by either of the two proposed mechanisms: enzyme bridged and restricted rotation.
Sikder
D
D
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
Nagaraja
V
V
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D019175:DNA Methylation; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D008958:Models, Molecular; D009161:Mycobacterium / Q000201:enzymology*; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000378:metabolism; D011196:Potassium Permanganate / Q000378:metabolism; D011485:Protein Binding; D012399:Rotation; D055672:Static Electricity; D013379:Substrate Specificity; D013463:Sulfuric Acid Esters / Q000378:metabolism
D004268:DNA-Binding Proteins; D010710:Phosphates; D013463:Sulfuric Acid Esters; D011196:Potassium Permanganate; D004247:DNA; D003850:Deoxyribonuclease I; D004264:DNA Topoisomerases, Type I; C007482:dimethyl sulfate