Preliminary X-ray crystallographic and NMR studies on the exonuclease domain of the epsilon subunit of Escherichia coli DNA polymerase III

J Struct Biol. 2000 Aug;131(2):164-9. doi: 10.1006/jsbi.2000.4291.

Abstract

The structured core of the N-terminal 3'-5' exonuclease domain of epsilon, the proofreading subunit of Escherichia coli DNA polymerase III, was defined by multidimensional NMR experiments with uniformly (15)N-labeled protein: it comprises residues between Ile-4 and Gln-181. A 185-residue fragment, termed epsilon(1-185), was crystallized by the hanging drop vapor diffusion method in the presence of thymidine-5'-monophosphate, a product inhibitor, and Mn(2+) at pH 5.8. The crystals are tetragonal, with typical dimensions 0.2 mm x 0.2 mm x 1.0 mm, grow over about 2 weeks at 4 degrees C, and diffract X-rays to 2.0 A. The space group was determined to be P4(n)2(1)2 (n = 0, 1, 2, 3), with unit cell dimensions a = 60.8 A, c = 111.4 A.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain*
  • Chymotrypsin / metabolism
  • Crystallization
  • Crystallography, X-Ray
  • DNA Polymerase III / chemistry*
  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / enzymology*
  • Exodeoxyribonucleases / chemistry*
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism
  • Manganese / metabolism
  • Mutagenesis
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Protein Structure, Tertiary
  • Protein Subunits
  • Sequence Alignment
  • Thymidine Monophosphate / metabolism

Substances

  • Peptide Fragments
  • Protein Subunits
  • Thymidine Monophosphate
  • Manganese
  • DNA Polymerase III
  • Exodeoxyribonucleases
  • Chymotrypsin