Function of the hydrophilic carboxyl terminus of type II DNA topoisomerase from Drosophila melanogaster. I. In vitro studies.
The function of the hydrophilic carboxyl-terminal region of Drosophila DNA topoisomerase II was examined by constructing a series of deletion mutants at the 3'-end of the Drosophila Top2 cDNA. The truncated enzymes were then expressed in Saccharomyces cerevisiae. Deletion of up to 240 out of 1447 total amino acids had no apparent effect on the enzyme's ability to catalyze topisomerization reactions. When 273, or more, amino acids were deleted, the enzyme was no longer active. Examples were found where deletion of less than 240 amino acids inactivated the enzyme. Based on the hydrodynamic properties determined for one of these mutants, the lack of activity was most likely due to misfolding of the polypeptides. The active mutants have similar hydrodynamic properties and heat inactivation profiles as the intact enzyme, suggesting that they are dimeric and stably folded. The carboxyl-terminal 240 amino acids also were not required for interaction with the drug VM26. The only difference noted between the shortest, active mutant and the full-length enzyme was a decrease in the stability of the interaction of the truncated enzyme with DNA as evidenced by a decrease in the ionic strength at which catalysis was optimal and at which the transition between a processive and distributive mode of supercoil relaxation occurred.
Crenshaw
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DG
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Hsieh
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded; D004331:Drosophila melanogaster / Q000201:enzymology*; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae; D017384:Sequence Deletion; D013713:Teniposide / Q000378:metabolism
D004277:DNA, Single-Stranded; D013713:Teniposide; D004250:DNA Topoisomerases, Type II