FIG. 4.

Effects of T-antigen-specific monoclonal antibodies on biochemical activities of T antigen. (A) T-antigen binding to a labeled SV40 origin DNA fragment was tested in a band shift in the presence of the indicated monoclonal antibodies (lanes 3 to 11), without antibody (lane 2), or without T antigen (lane 1). (B) ATPase reactions were carried out without T antigen (lane 1), with T antigen (lane 2), or with T antigen in the presence of the indicated monoclonal antibody (lanes 3 to 11) or buffer (lane 12). The reaction products were separated by ascending thin-layer chromatography. Helicase reactions were performed with M13 DNA annealed to a labeled primer (C), and unwinding reactions were performed with a labeled duplex origin DNA fragment (ori) and a labeled nonspecific DNA fragment (ns) (D), in the presence of the indicated monoclonal antibodies (lanes 3 to 11) or buffer (lanes 12). Negative control reactions were performed without T antigen (lanes 1); positive controls were performed with T antigen and without antibodies (lanes 2) as indicated. (C and D) The substrate DNA in the native conformation (lane N) or after heat denaturation (lane D) was electrophoresed in parallel. Quantitative evaluation of the autoradiograms for each reaction is given below each lane ss and ds, single-stranded and double-stranded DNA, respectively. (E) SV40 DNA unwinding assays contained closed circular supercoiled pUC-HS DNA, T antigen (TAg; lanes 2 to 10), topoisomerase I, and E. coli SSB. Reactions were carried out in the presence of monoclonal antibodies as indicated (lanes 4 to 10) or antibody buffer (lane 3). Reaction products were analyzed by electrophoresis and ethidium bromide staining. Form U, underwound covalently closed circular DNA.