Effect of 2′-5′ Phosphodiesters on DNA Transesterification by Vaccinia Topoisomerase

Berit O. Krogh, Christopher D. Claeboe, Sidney M. Hecht, Stewart Shuman

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Vaccinia topoisomerase forms a covalent DNA-(3′ -phosphotyrosyl)-enzyme intermediate at a pentapyri-midine target site 5′-CCCTTp ↓ in duplex DNA. By introducing single 2′-5′ phosphodiesters in lieu of a standard 3′-5′ phosphodiester linkage, we illuminate the contributions of phosphodiester connectivity to DNA transesterification. We find that the DNA cleavage reaction was slowed by more than six orders of magnitude when a 2′-5′ linkage was present at the scissile phosphodiester (CCCTT2′p ↓ 5′A). Thus, vaccinia topoisomerase is unable to form a DNA-(2′-phosphotyrosyl)-enzyme intermediate. We hypothesize that the altered geometry of the 2′-5′ phosphodiester limits the ability of the tyrosine nucleophile to attain a requisite, presumably apical orientation with respect to the 5′-OH leaving group. A 2′-5′ phosphodiester located to the 3′ side of the cleavage site (CCCTTp ↓ N2′ p5′N) reduced the rate of transesterification by a factor of 500. In contrast, 2′-5′ phosphodiesters at four other sites in the scissile strand (TpCGCCCTpT ↓ ATpTpC) and five positions in the nonscissile strand (3′-GGGpApApTpApA) had no effect on transesterification rate. The DNAs containing 2′-5′ phosphodiesters were protected from digestion by exonuclease III. We found that exonuclease III was consistently arrested at positions 1 and 2 nucleotides prior to the encounter of its active site with the modified 2′-5′ phosphodiester and that the 2′-5′ linkage itself was poorly hydrolyzed by exonuclease III.

Original languageEnglish (US)
Pages (from-to)20907-20912
Number of pages6
JournalJournal of Biological Chemistry
Volume276
Issue number24
DOIs
StatePublished - Jun 15 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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