FIG. 1.

Comparison of the activities of topo I isolated from different species. (A) In vitro DNA replication assays were performed with topo I-deficient 293 cell extracts supplemented with 200 ng of pSKori, 1 μCi of [α-³²P]dATP, and 2 μg of T antigen in the absence (lane 2) or presence of increasing amounts of human, bovine, or E. coli topo I (50 ng [lane 3, 7, or 11], 100 ng [lane 4, 8, or 12], 200 ng [lane 5, 9, or 13], and 300 ng [lane 6, 10, or 14]. As a control, neither T antigen nor topo I was added to lane 1. After purification, the labeled replicated DNA was analyzed by gel electrophoresis on a 1.5% agarose gel. The positions of RIs, CCC relaxed DNA, and form I DNAs are shown. (B) ELISAs were performed by adsorbing 100 ng of human, bovine (CT), or E. coli topo I to the wells of a microtiter plate and reacting them with increasing quantities (0, 0.75, 1.5, 3, 6.25, 12.5, 25, 50, 100, and 200 ng) of T antigen. The specific complexes formed were detected by using biotinylated PAb101, streptavidin-conjugated HRP, and o-phenylenediamine substrate solution. Absorbance at (optical density [OD]) 490 nm was measured with a Dynex MRX plate reader.