Open AccessAn activity gel assay for the detection of DNA helicases and nucleases from cell-free extracts
Author(s) -
Sanjay K. Shukla,
David McCarthy
Publication year - 1994
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/22.9.1626
Subject(s) - helicase , biology , dna , microbiology and biotechnology , exonuclease , gel electrophoresis , prokaryotic dna replication , rna helicase a , primase , endonuclease , recq helicase , biochemistry , circular bacterial chromosome , dna replication , dna polymerase , gene , polymerase chain reaction , rna , reverse transcriptase
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.
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