Zinc (II) coordination in Escherichia coli DNA topoisomerase I is required for cleavable complex formation with DNA.
Escherichia coli DNA topoisomerase I catalyzes relaxation of negatively supercoiled DNA. The reaction proceeds through a covalent intermediate, the cleavable complex, in which the DNA is cleaved and the enzyme is linked to the DNA via a phosphotyrosine linkage. Each molecule of E. coli DNA topoisomerase I has been shown to have three tightly bound zinc(II) ions required for relaxation activity (Tse-Dinh, Y.-C., and Beran-Steed, R.K. (1988) J. Biol. Chem. 263, 15857-15859). It is shown here that Cd(II) could replace Zn(II) in reconstitution of active enzyme from apoprotein. The role of metal was analyzed by studying the partial reactions. The apoenzyme was deficient in sodium dodecyl sulfate-induced cleavage of supercoiled PM2 phage DNA. Formation of covalent complex with linear single-stranded DNA was also reduced in the absence of metal. However, the cleavage of small oligonucleotide was not affected, and the apoenzyme could religate the covalently bound oligonucleotide to another DNA molecule. Assay of noncovalent complex formation by retention of 5'-labeled DNA on filters showed that the apoenzyme was not inhibited in noncovalent binding to DNA. It is proposed that zinc(II) coordination in E. coli DNA topoisomerase I is required for the transition of the noncovalent complex with DNA to the cleavable state.
Tse-Dinh
Y C
YC
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla 10595.
D000595:Amino Acid Sequence; D001345:Autoradiography; D002104:Cadmium / Q000378:metabolism; D002384:Catalysis; D002614:Chelating Agents; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D015032:Zinc / Q000378:metabolism*
D002614:Chelating Agents; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D002104:Cadmium; D004264:DNA Topoisomerases, Type I; D015032:Zinc