DNA Recombination pp 329-343

Part of the Methods in Molecular Biology book series (MIMB, volume 745)

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Quality Control of Purified Proteins Involved in Homologous Recombination

Protocol

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.

Key words

ATPase DNA helicase DNA translocase endonuclease exonuclease phosphatase topoisomerase in vitro recombination assays protein purification 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of MicrobiologyUniversity of CaliforniaDavisUSA
  2. 2.Department of Microbiology and Department of Molecular and Cellular BiologyUniversity of CaliforniaDavisUSA

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