Enhanced binding to origin DNA at low pH enables easy detection of polyomavirus large T antigen by gel mobility shift assay of unfixed complexes.
Enhanced, stable binding by polyomavirus large T antigen to the viral DNA replication origin at pH 6 allowed the development of a gel mobility shift assay for the detection of large T antigen. Such assays were not possible at pH 7.6 without previous fixation, due to instability of the complexes. We demonstrated that the gel mobility shift assay at pH 6 is very sensitive, allowing the detection of as little as 5 ng large T antigen, and is highly specific for DNA containing G(A/G)GGC target sequences. This method was used to detect large T antigen in crude cell lysates from transformed yeast cell lines or nuclear extracts from infected insect cells. Large T antigen-DNA complexes remained at or near the loading well in 5% acrylamide or 1.5% agarose gels, indicating that these complexes are very large. Glycerol gradient analysis showed that protein-DNA complexes formed at pH 6 were massive, and that large T antigen also formed large complexes when incubated at low pH in the absence of DNA. These results show that pH has a major effect on binding of large T antigen to its multiple target sites in the viral origin of DNA replication, presumably by affecting protein-protein interactions that are important for the stability of large T antigen-DNA complexes.
Peng
Y C
YC
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Acheson
N H
NH
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D004279:DNA, Viral / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D018741:Replication Origin; D012680:Sensitivity and Specificity
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D011994:Recombinant Proteins