PURIFICATION, CRYSTALLIZATION AND SPACE GROUP DETERMINATION OF DNA-REPAIR ENZYME EXONUCLEASE-III FROM ESCHERICHIA-COLI

Escherichia coli exonuclease III possesses multiple catalytic activities: (1) a nucleotidyl hydrolase activity cutting 5’ to apurinic/apyrimidinic sites and urea residues in DNA; (2) a 3’ to 5’ exonuclease activity specific for double-stranded DNA; (3) a RNase H activity preferentially degrading the RNA strand of a DNA·RNA hybrid and (4) an activity that can remove a number of 3’ termini from duplex DNA including 3’ phosphates, 3’ phosphoglycolate residues, 3’ phosphoglycolaldehyde residues and 3’ trans-4-hydroxy-2-pentenal-5-phosphate residues. These multiple activities make exonuclease III a major enzyme in the base excision repair pathway for DNA damage. We have purified exonuclease III and grown crystals by the vapor diffusion method using polyethylene glycol 4000 as the precipitant. Buffers were found to have profound effects on crystallization with high concentrations of imidazole/malate buffer (0.4 M to 1.0 M) yielding larger crystals with less twinning. The crystals belong to the space group P3121 or its enantiomorph P3221 with unit cell dimensions of a = b = 107.8 AÅ, c = 42.2 AÅ, α = β = 90°, γ = 120°, have one 31 kDa monomer per asymmetric unit and diffract to 1.6 AÅ. These crystals are stable to X-rays and suitable for high resolution structure determination. © 1993 Academic Press, Inc.