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Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.
|M13mp18 single strand DNA (250 ng/µl)||4 µl|
|Oligo (35ng/µl)||1 µl|
|0.1 MTris-Cl (pH7.6)||4.3 µl||10 mM|
|10 mM MgCl2||4.3 µl||1 mM|
|Add H2O to final volume 43 µl.|
|10× helicase assay buffer||5 µl|
|100 mM ATP||1 µl|
|Stock||20 ml||Final conc.|
|1M Tris-Cl [pH 8.0]||200 µl||10 mM|
|5M NaCl||1600 µl||400 mM|
|0.1 M PMSF||40 µl||0.2 mM|
|1 mg/ml Aprotinin||20 µl||1 µg/ml|
|1 mg/ml Leupeptin||20 µl||1 µg/ml|
|1 mg/ml Pepstatin A||20 µl||1 µg/ml|
|ddH2O to 20 ml|
|Stock||10 ml||10× Final conc.|
|1 M Tris-Cl (pH 7.5)||2 ml||200 mM|
|1 M MgCl2||1 ml||100 mM|
|10 mg/ml BSA||1 ml||1 mg/ml|
|Acrylamide||Acrylamide (30%, 29:1)|
This protocol is adapted from our previous publication Li et al. (2012). We acknowledge the insightful critiques of the manuscript from members from our laboratory. This study was supported by the HIV-Associated Malignancies Pilot Project Award (National Cancer Institute), National Institutes of Health (NIH) grants R01CA148768 and R01CA142723, and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
1Addition of 10% of glycerol in IPP400 buffer is helpful but not necessary.
2Immoblizing protein on other resins may also work, but the authors have not tested other resin systems.
3Freezing-and-thawing labeled substrates will bring unexpected bands.
4If you want to get rid of free labeled nucleic acid from the oligo substrate using ethenol precipitation, DNA purification kits, etc, the quality of purified substrate should be monitored to make sure the labeled short oligo is not dissociated from the M13mp18 genome.
5If alernative single-strand DNA, DNA oligos or radiolabeled deoxyribonucleotides (such as [α-32P] dCTP, etc.) are used, the addition of free deoxyribonucleotides in step B7 is adjustable. Noticably, your design should make sure that your radiolabeled deoxyribonucleotides have been incorporated into your substrates before the reaction is stopped by deoxyribonucleotides deficiency (In this protocol, the reaction will be stopped by dTTP deficiency.).