Site-specific DNA transesterification by vaccinia topoisomerase: Role of specific phosphates and nucleosides

Chonghui Cheng, Stewart Shuman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-CCCTTp↓ in duplex DNA. Here we present experiments that illuminate the contributions of specific nucleosides and phosphates to site affinity and transesterification. We find that the -1 phosphate and -2 nucleoside on the scissile strand (5'- CCCTTp↓NpN) enhance the rate of transesterification by factors of 40 and 25, respectively, whereas the DNA segment downstream of the -2 nucleotide makes no significant kinetic contribution. Placement of a 5'-phosphate/3'-OH nick at position +2, +3, +4, or +5 within the CCCTT element results in a 5-10-fold reduction in the affinity of topoisomerase binding to DNA. A nick at the +2 phosphate also slows the rate of transesterification by ~500-fold. This finding, together with earlier studies of the effects of position-specific base and sugar modifications, points to the +2 Tp nucleotide as being the most critical element of the CCCTT target site other than the scissile phosphate itself. On the noncleaved strand, the segment downstream of the 3'- GGGAA element contributes minimally to the rate of transesterification provided that the substrate is otherwise fully base-paired within the 5'- CCCTT target site. By studying the effects of single nucleotide gaps and missing phosphate nicks within the 3'-GGGAA sequence, we find that the +1 and +2 adenosine nucleosides enhance the rate of transesterification by 20- and 1000-fold respectively and that the +5 phosphate (3'-GpGGAA) is also important for cleavage. Cumulative functional analyses of the vaccinia topoisomerase-DNA interface are discussed in light of newly available structures for the vaccinia and human type IB enzymes.

Original languageEnglish (US)
Pages (from-to)16599-16612
Number of pages14
JournalBiochemistry
Volume38
Issue number50
DOIs
StatePublished - Dec 14 1999

ASJC Scopus subject areas

  • Biochemistry

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