title: An activity gel assay for the detection of DNA helicases and nucleases from cell-free extracts.
abstract: An activity gel assay was developed for the detection of DNA helicases in crude extracts. The assay was based on the ability of DNA helicases to unwind radioactive fragments from single-stranded M13 circles that were immobilized in an SDS polyacrylamide gel. The displaced radioactive strands were detected by blotting them to a filter and visualizing the resulting bands by autoradiography. Experiments with purified proteins demonstrated that DNA helicases, endonucleases and exonucleases could produce activity bands. A one-dimensional gel assay was sufficiently sensitive to allow detection of DNA helicase I, DNA helicase II, DNA helicase IV, the RecQ helicase as well as 3 unidentified putative DNA helicases in crude extracts of Escherichia coli. Exonuclease and endonuclease activities from crude extracts could be distinguished from DNA helicase activities by their ATP-independence and from each other by their band morphologies.
authors.affiliation: Department of Botany and Microbiology, University of Oklahoma, Norman 73019-0245.
authors.forename: S K
authors.initials: SK
authors.lastname: Shukla
authors.forename: D
authors.initials: D
authors.lastname: McCarthy
mesh_terms: D001483:Base Sequence; D002474:Cell-Free System; D004265:DNA Helicases / Q000032:analysis*; D017931:DNA Primers; D003851:Deoxyribonucleases / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data
chemical_list: D017931:DNA Primers; D003851:Deoxyribonucleases; D004265:DNA Helicases