Remote Phosphate Contacts Trigger Assembly of the Active Site of DNA Topoisomerase IB

Ligeng Tian, Christopher D. Claeboe, Sidney Hecht, Stewart Shuman

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Vaccinia topoisomerase IB forms a covalent DNA-(3′-phosphotyrosyl)- enzyme intermediate at its target site 5′-CCCTTp↓ in duplex DNA. The contributions of backbone electrostatics and individual phosphate oxygens to the transesterification reaction were probed by introducing 22 single Rp and Sp methylphosphonate diastereomers at 11 positions flanking the cleavage site. Methyl groups at eight positions (four on the scissile strand and four on the nonscissile strand) inhibited the rate of single-turnover cleavage by factors of 50-50,000. Stereospecific interference was observed at several phosphates, thereby distinguishing simple electrostatic contributions from putative specific polar contacts to either the pro-Sp or pro-Rp oxygens. The functionally relevant phosphate oxygens are located on the minor groove face of the helix on which the scissile phosphodiester resides. Our findings, combined with available crystal structures of vaccinia and human topoisomerase IB, show how specific phosphate contacts remote from where chemistry occurs are critical for assembly of the active site.

Original languageEnglish (US)
Pages (from-to)31-40
Number of pages10
JournalStructure
Volume12
Issue number1
DOIs
StatePublished - Jan 2004
Externally publishedYes

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Type I DNA Topoisomerase
Catalytic Domain
Phosphates
Vaccinia
Oxygen
Static Electricity
DNA
Enzymes

ASJC Scopus subject areas

  • Molecular Biology
  • Structural Biology

Cite this

Remote Phosphate Contacts Trigger Assembly of the Active Site of DNA Topoisomerase IB. / Tian, Ligeng; Claeboe, Christopher D.; Hecht, Sidney; Shuman, Stewart.

In: Structure, Vol. 12, No. 1, 01.2004, p. 31-40.

Research output: Contribution to journalArticle

Tian, Ligeng ; Claeboe, Christopher D. ; Hecht, Sidney ; Shuman, Stewart. / Remote Phosphate Contacts Trigger Assembly of the Active Site of DNA Topoisomerase IB. In: Structure. 2004 ; Vol. 12, No. 1. pp. 31-40.
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