Cocrystal structure of an editing complex of Klenow fragment with DNA

Proc Natl Acad Sci U S A. 1988 Dec;85(23):8924-8. doi: 10.1073/pnas.85.23.8924.

Abstract

High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydrophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3' terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus. Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Computer Simulation
  • DNA / metabolism*
  • DNA Polymerase I / metabolism*
  • DNA, Single-Stranded / metabolism
  • Models, Molecular
  • Nucleic Acid Conformation
  • Protein Binding
  • Protein Conformation
  • X-Ray Diffraction

Substances

  • DNA, Single-Stranded
  • DNA
  • DNA Polymerase I