Nucleic Acids Research, Vol 25, Issue 18 3649-3656, Copyright © 1997 by Oxford University Press
J Sekiguchi and S Shuman
Vaccinia DNA topoisomerase catalyzes the cleavage and re-joining of DNA
strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a
specific target sequence, 5'-(C/T)CCTT downward arrow. The 314 aa protein
consists of three protease-resistant structural domains demarcated by
protease-sensitive interdomain segments referred to as the bridge and the
hinge. The bridge is defined by trypsin-accessible sites at Arg80, Lys83
and Arg84. Photocrosslinking and proteolytic footprinting experiments
suggest that residues near the interdomain bridge interact with DNA. To
assess the contributions of specific amino acids to DNA binding and
transesterification chemistry, we introduced alanine substitutions at 16
positions within a 24 aa segment from residues 63 to
86(DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation under
conditions optimal for the wild-type topoisomerase revealed significant
mutational effects at six positions; Arg67, Tyr70, Tyr72, Arg80, Arg84 and
Asp85. The mutated proteins displayed normal or near-normal rates of
single-turnover transesterification to DNA. The effects of amino acid
substitutions on DNA binding were evinced by inhibition of covalent adduct
formation in the presence of salt and magnesium. The mutant enzymes also
displayed diminished affinity for a subset of cleavage sites in pUC19 DNA.
Tyr70 and Tyr72 were subjected to further analysis by replacement with Phe,
His, Gln and Arg. At both positions, the aromatic moiety was important for
DNA binding.
ARTICLES
Mutational analysis of vaccinia virus topoisomerase identifies residues involved in DNA binding
Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA.
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