Chemical and traditional mutagenesis of vaccinia DNA topoisomerase provides insights to cleavage site recognition and transesterification chemistry.

ABSTRACT: Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate. Here we gained new insights to the TopIB mechanism through "chemical mutagenesis." Meta-substituted analogs of Tyr(274) were introduced by in vitro translation in the presence of a chemically misacylated tRNA. We report that a meta-OH reduced the rate of DNA cleavage 130-fold without affecting the rate of religation. By contrast, meta-OCH(3) and NO(2) groups elicited only a 6-fold decrement in cleavage rate. We propose that the meta-OH uniquely suppresses deprotonation of the para-OH nucleophile during the cleavage step. Assembly of the vaccinia TopIB active site is triggered by protein contacts with a specific DNA sequence 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p

SUBMITTER: Yakovleva L 

PROVIDER: S-EPMC2414267 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature