Development of an activity gel assay for DNA metabolic proteins and evidence that Escherichia coli ribosomal protein S2 has an endodeoxyribonuclease activity

A DNA helicase activity gel assay was developed which allows detection of helicases and other DNA metabolic enzymes based on their abilities to release immobilized radioactive DNA from a polyacrylamide gel. Released DNAs are blotted to a membrane and detected by autoradiography. The assay can also be used to detect nucleases, restriction enzymes and single stranded DNA binding (Ssb) proteins. The activity bands from the last two classes of enzymes have been shown only with purified enzymes. DNA helicase and nuclease activities can be distinguished from each other on the basis of ATP-dependence. Helicases produce activity bands only in the presence of ATP. Endonucleases produce ATP-independent dark bands whereas exonucleases give clear (lighter than background) bands which are also ATP-independent. Restriction enzymes and Ssb also give ATP-independent dark bands. Four of the 10 known E. coli helicases (Helicase I, Helicase II, Helicase IV, and Rec Q helicase) have been detected from crude extracts with the use of the activity gel assay. Two previously unidentified ATP-dependent activities presumably represent newly discovered DNA helicases. Rep helicase activity has been detected with purified protein although an ATP-dependent activity with the same electrophoretic mobility as Rep has been detected in crude extracts. Most of the undetected helicases (RecB, PriA, DnaB, helicase III, and uvrA/B) can be potentially detected with modified activity gel substrates. For example, a branched partial duplex DNA should allow detection of DnaB helicase. Taking into account the properties of the known E. coli DNA metabolic enzymes, 38 activities were expected to be detected. A two-dimensional helicase activity gel assay revealed 68 activity bands. Therefore it appears that a relatively large number of E. coli DNA metabolic genes remain to be identified.;The activity gel assay was used to purify 2 major apparent DNases from E. coli with mobilities corresponding to 26 kDa and 24 kDa. N-terminal sequences of these partially purified proteins were determined and were found to correspond ribosomal proteins S2 and S3 respectively. The association of a nonspecific endonuclease activity was confirmed with purified S2 in a conventional assay. The apparent exonuclease activity band of S3 was due to its ability to bind to single-stranded DNA. S3-DNA complexes were apparently trapped in the activity gel and failed to contribute to the background in the activity gel filter. Possible roles of ribosomal proteins in DNA metabolism are considered.;The helicase activity gel assay was optimized to detect DNA metabolic enzymes from human cell lines. The modified assay could then be used as a screening tool to detect DNA metabolic defects in patients who are defective for DNA repair or who are hypersusceptible to cancer. Unlike in the case of E. coli, I detected more activity bands from human cell extracts in high percentage (...