title: Quality control of purified proteins involved in homologous recombination.
abstract: Biochemical reconstitution using purified proteins and defined DNA substrates is a key approach to develop a mechanistic understanding of homologous recombination. The introduction of sophisticated purification tags has greatly simplified the difficult task of purifying individual proteins or protein complexes, generating a wealth of mechanistic information. Using purified proteins in reconstituted recombination assays necessitates strict quality control to eliminate the possibility that relevant protein or nucleic acid contaminations lead to misinterpretation of experimental data. Here we provide simple protocols that describe how to detect in purified protein preparations contaminating nucleic acids and relevant enzymatic activities that may interfere with in vitro recombination assays. These activities include ATPases, indicating the potential presence of helicases or translocases, endo- and exonucleases, phosphatases, and type I or type II topoisomerases.
authors.affiliation: Department of Microbiology, University of California, 95616-8665, Davis, CA 95616-8665, USA. xpzhang@ucdavis.edu
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Zhang
authors.forename: Wolf-Dietrich
authors.initials: WD
authors.lastname: Heyer
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D002855:Chromatography, Thin Layer; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*
chemical_list: D004720:Endonucleases; D005092:Exonucleases; D010744:Phosphoric Monoester Hydrolases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II