Purification and characterization of RecQ helicases of plants.
Helicases are essential for DNA metabolism. Different helicases have different properties tailored to fulfill their specific tasks. RecQ-helicases are known to be important in DNA repair and DNA recombination. In higher organisms several RecQ homologues can be identified. For instance, seven RecQ homologues were identified in the model plant Arabidopsis thaliana. Specialization of those proteins can possibly be reflected by differences in their biochemical substrate spectrum. Moreover, a helicase of interest might be defined by its biochemical properties as a functional ortholog of a RecQ helicase in other organisms. In this chapter the initial steps that will provide the basis for a proper biochemical characterization are given. After the description of the expression of the helicase of interest in the heterologous host Escherichia coli, its purification with the help of two affinity tags and the preparation of a model DNA substrate for the strand displacement assay are described. Finally, it is shown how this model substrate can be used to ensure the purity of the enzymatic preparation of interest.
Botanik II, UniversitAt Karlsruhe (TH), Karlsruhe, Germany.
Daniela
D
Kobbe
Manfred
M
Focke
Holger
H
Puchta
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053484:RecQ Helicases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D029681:Arabidopsis Proteins; D002147:Calmodulin; D011994:Recombinant Proteins; D004247:DNA; D053484:RecQ Helicases