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Crystallization and crystallographic characterization of the iron sulfur-containing DNA-repair enzyme endonuclease-iii from escherichia-coli

Academic Article
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Overview

authors

  • Kuo, C. F.
  • McRee, Duncan
  • Cunningham, R. P.
  • Tainer, John

publication date

  • September 1992

journal

  • Journal of Molecular Biology  Journal

abstract

  • Endonuclease III from Escherichia coli is an iron-sulfur enzyme possessing both DNA N-glycosylase and apurinic/apyrimidinic lyase activities. It could serve to repair damaged thymine residues in DNA via base excision-repair. We have crystallized endonuclease III by a combination of dialysis and seeding techniques after exploration of a wide variety of precipitants which failed to yield macroscopic crystals. Important features of the optimized crystallization include: the use of 5 to 10% glycerol, a temperature of 15 degrees C, controlled dialysis to decrease ionic strength and macroseeding using a 200 mM-NaCl transfer buffer to dissolve microcrystalline contamination. The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 48.5 A, b = 65.8 A, c = 86.8 A, alpha = beta = gamma = 90 degrees, have one 23 kDa monomer per asymmetric unit, and diffract to 1.84 A. A native anomalous Patterson map located the iron-sulfur cluster and reaffirmed its existence. The reported crystallization procedures ensure an ample supply of crystals for the extensive heavy-atom derivative search necessary for this labile iron-sulfur enzyme. The elucidation of endonuclease III structure will facilitate not only the understanding of glycosylase and lyase mechanisms but also the structure and function of this new class of iron-sulfur proteins.

subject areas

  • Bacterial Proteins
  • Crystallography
  • DNA Repair
  • Deoxyribonuclease (Pyrimidine Dimer)
  • Endodeoxyribonucleases
  • Escherichia coli
  • Escherichia coli Proteins
  • Iron-Sulfur Proteins
  • Protein Conformation
  • Recombinant Proteins
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Research

keywords

  • CRYSTALLIZATION
  • DNA EXCISION-REPAIR
  • ENDONUCLEASE-III
  • IRON SULFUR PROTEIN
  • X-RAY DIFFRACTION
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Identity

International Standard Serial Number (ISSN)

  • 0022-2836

Digital Object Identifier (DOI)

  • 10.1016/0022-2836(92)90703-m

PubMed ID

  • 1522598
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Additional Document Info

start page

  • 347

end page

  • 351

volume

  • 227

issue

  • 1

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