Cloning, expression, purification and characterization of DNA topoisomerase I of Mycobacterium tuberculosis.
The complete gene encoding Topoisomerase 1 (Topo I) from Mycobacterium tuberculosis (MTb), Erdman strain, has been isolated and sequenced. The coding region of this gene is 2700 nt encoding a polypeptide of 900 amino acids with a calculated molecular mass of 99353 Da. The amino-acid sequence identity compared to E. coli and Synechococcus Topo I is 22 and 30%, respectively. The gene was expressed in E. coli BL21(DE3) and purified to near homogeneity. Recombinant MTb Topo I is enzymatically active, relaxing negatively supercoiled DNA in a magnesium-dependent, ATP-independent reaction. Spermidine, a typical inhibitor of prokaryotic type I DNA topoisomerase, inhibits the activity. Unlike the more well-characterized E. coli Topo I, MTb Topo I does not contain a zinc-finger DNA-binding motif in the C-terminal domain of the protein.
Yang
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F
Department of Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Lu
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Rubin
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H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D004264:DNA Topoisomerases, Type I