Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates

Chad A Brautigam, Thomas A. Steitz

Research output: Contribution to journalArticlepeer-review

156 Scopus citations

Abstract

A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 Å resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the R(p) isomer of phosphorothioate DNA, refined at 2.2 Å resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the S(p) phosphorothioate at 2.3 Å resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the R(p) isomer was reduced only about 15-fold, while no enzyme activity could be detected with the S(p) phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the S(p) phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments.

Original languageEnglish (US)
Pages (from-to)363-377
Number of pages15
JournalJournal of Molecular Biology
Volume277
Issue number2
DOIs
StatePublished - Mar 27 1998

Keywords

  • 3'-5' exonuclease
  • Mn rescue
  • Phosphorothioates
  • Two-metal-ion mechanism
  • X-ray crystallography

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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