Open AccessCrystallization and preliminary X‐ray characterization of two thermostable DNA nucleases
Author(s) -
Kuettner E. Bartholomeus,
Pfeifer Sven,
Keim Antje,
GreinerStöffele Thomas,
Sträter Norbert
Publication year - 2006
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309106050548
Subject(s) - exonuclease iii , exonuclease , escherichia coli , dna , enzyme , chemistry , biochemistry , oligonucleotide , biology , gene , dna polymerase
Temperature‐tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA‐cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4‐methyl pentandiol, dioxane, ethanol or 2‐propanol as precipitants. The addition of a 10‐mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes.