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  • Title: Crystallization and preliminary crystallographic analysis of endonuclease VIII in its uncomplexed form.
    Author: Golan G, Zharkov DO, Fernandes AS, Zaika E, Kycia JH, Wawrzak Z, Grollman AP, Shoham G.
    Journal: Acta Crystallogr D Biol Crystallogr; 2004 Aug; 60(Pt 8):1476-80. PubMed ID: 15272182.
    Abstract:
    The Escherichia coli DNA repair enzyme endonuclease VIII (EndoVIII or Nei) excises oxidized pyrimidines from damaged DNA substrates. It overlaps in substrate specificity with endonuclease III and may serve as a back-up for this enzyme in E. coli. The three-dimensional structure of Nei covalently complexed with DNA has been recently determined, revealing the critical amino-acid residues required for DNA binding and catalytic activity. Based on this information, several site-specific mutants of the enzyme have been tested for activity against various substrates. Although the crystal structure of the DNA-bound enzyme has been fully determined, the important structure of the free enzyme has not previously been analyzed. In this report, the crystallization and preliminary crystallographic characterization of DNA-free Nei are described. Four different crystal habits are reported for wild-type Nei and two of its catalytic mutants. Despite being crystallized under different conditions, all habits belong to the same crystal form, with the same space group (I222) and a similar crystallographic unit cell (average parameters a = 57.7, b = 80.2, c = 169.7 A). Two of these crystal habits, I and IV, appear to be suitable for full crystallographic analysis. Crystal habit I was obtained by vapour diffusion using PEG 8000, glycerol and calcium acetate. Crystal habit IV was obtained by a similar method using PEG 400 and magnesium chloride. Both crystals are mechanically strong and stable in the X-ray beam once frozen under cold nitrogen gas. A full diffraction data set has recently been collected from a wild-type Nei crystal of habit I (2.6 A resolution, 85.2% completeness, Rmerge = 9.8%). Additional diffraction data were collected from an Nei-R252A crystal of habit IV (2.05 A resolution, 99.9% completeness, Rmerge = 6.0%) and an Nei-E2A crystal of habit IV (2.25 A resolution, 91.7% completeness, Rmerge = 6.2%). These diffraction data were collected at 95-100 K using a synchrotron X-ray source and a CCD area detector. All three data sets are currently being used to obtain crystallographic phasing via molecular-replacement techniques.
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