Replacement of the active site tyrosine of vaccinia DNA topoisomerase by glutamate, cysteine or histidine converts the enzyme into a site-specific endonuclease.
Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5'-CCCTT downward arrow sites in duplex DNA. The T downward arrow nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that mutant enzymes containing glutamate, cysteine or histidine in lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp duplex DNA at the CCCTT downward arrow site to yield a 3' phosphate-terminated product. The Cys-274 mutant forms trace levels of a covalent protein-DNA complex, suggesting that the DNA cleavage reaction may proceed through a cysteinyl-phosphate intermediate. However, the His-274 and Glu-274 mutants evince no detectable accumulation of a covalent protein-DNA adduct. Glu-274 is the most active of the mutants tested. The pH dependence of the endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that of the wild-type enzyme in hydrolysis of the covalent adduct (optimum pH = 9.5). At pH 6.5, the Glu-274 endonuclease reaction is slower by 5-6 orders of magnitude than the rate of covalent adduct formation by the wild-type topoisomerase, but is approximately 20 times faster than the rate of hydrolysis by the wild-type covalent adduct. We discuss two potential mechanisms to account for the apparent conversion of a topoisomerase into an endonuclease.
Wittschieben
J
J
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Petersen
B O
BO
Shuman
S
S
D001665:Binding Sites; D003545:Cysteine; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D018698:Glutamic Acid; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D016296:Mutagenesis; D013329:Structure-Activity Relationship; D014443:Tyrosine; D014616:Vaccinia virus / Q000201:enzymology*
D004278:DNA, Superhelical; D018698:Glutamic Acid; D014443:Tyrosine; D006639:Histidine; D004247:DNA; D004720:Endonucleases; D004264:DNA Topoisomerases, Type I; D003545:Cysteine