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

Lyudmila Yakovleva, Shengxi Chen, Sidney M. Hecht, Stewart Shuman

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14 Scopus citations

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 Tyr274 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-OCH3 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 +5C+4C+3T+2T+1p ↓ N (where ↓ denotes the cleavage site). A signature α-helix of the poxvirus TopIB (132GKMKYLKENETVG144) engages the target site in the major groove and thereby recruits catalytic residue Arg 130 to the active site. The effects of 11 missense mutations at Tyr136 highlight the importance of van der Waals interactions with the 3′-G+4pG+3p dinucleotide of the nonscissile strand for DNA cleavage and supercoil relaxation. Asn140 and Thr 142 donate hydrogen bonds to the pro-(Sp)-oxygen of the G+3pA+2 phosphodiester of the nonscissile strand. Lys 133 and Lys135 interact with purine nucleobases in the major groove. Whereas none of these side chains is essential per se, an N140A/T142A double mutation reduces the rate of supercoil relaxation and DNA cleavage by 120- and 30-fold, respectively, and a K133A/K135A double mutation slows relaxation and cleavage by 120- and 35-fold, respectively. These results underscore functional redundancy at the TopIB-DNA interface.

Original languageEnglish (US)
Pages (from-to)16093-16103
Number of pages11
JournalJournal of Biological Chemistry
Volume283
Issue number23
DOIs
StatePublished - Jun 6 2008

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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