An activity gel assay for the detection of DNA helicases and nucleases from cell-free extracts.
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.
Shukla
S K
SK
Department of Botany and Microbiology, University of Oklahoma, Norman 73019-0245.
McCarthy
D
D
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
D017931:DNA Primers; D003851:Deoxyribonucleases; D004265:DNA Helicases