Quality Control of Purified Proteins Involved in Homologous Recombination
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.
Key wordsATPase DNA helicase DNA translocase endonuclease exonuclease phosphatase topoisomerase in vitro recombination assays protein purification
We thank Clare Fasching, Erin Schwartz, Kirk Ehmsen, Shannon Ceballos, and William Wright for helpful comments on the manuscript. Our work is supported by the NIH (GM58015, CA92276), the DoD (BC083684), and a Susan G. Komen Breast Cancer Foundation postdoctoral fellowship (PDF403213) to XPZ.
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