Intradimerically tethered DNA topoisomerase II is catalytically active in DNA transport.
A covalently cross-linked dimer of yeast DNA topoisomerase II was created by fusing the enzyme with the GCN4 leucine zipper followed by two glycines and a cysteine. Upon oxidation of the chimeric protein, a disulfide bond forms between the two carboxyl termini, covalently and intradimerically cross-linking the two protomers. In addition, all nine of the cysteines naturally occurring in topoisomerase II have been changed to alanines in this construct. This cross-linked, cysteine-less topoisomerase II is catalytically active in DNA duplex passage as indicated by ATP-dependent DNA supercoil relaxation and kinetoplast DNA decatenation assays. However, these experiments do not directly distinguish between a "one-gate" and a "two-gate" mechanism for the enzyme.
Lindsley
J E
JE
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, 84132, USA.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D016831:Crithidia fasciculata; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D018105:DNA, Kinetoplast / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D018105:DNA, Kinetoplast; D004278:DNA, Superhelical; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II