Nucleic Acids Research, 2005, Vol. 33, No. 12 3815 Figure 2. Inhibition of SV40 T antigen helicase by DNA-bound mtDBP. (A and C) Schematic representation of DBP.For and DBP.Rev partial duplex substrates used in the unwinding reactions. The 46mer oligonucleotides O-DBP.for and O-DBP.rev containing the mtDBP binding sequence (underlined) were end- radiolabelled and annealed to the recombinant single-stranded DNA to construct the double-stranded region of DBP.For and DBP.Rev substrates, respectively. Numbers give the position of sea urchin mtDNA nucleotides; open arrow marks the orientation of the protein target site with respect to T antigen unwinding direction (black arrow). (B and D) Autoradiograms of representative displacement analyses showing the annealed partial duplex substrate (upper band) and the released oligonucleotide (lower band). Assays were performed as described in Materials and Methods, in the presence of 5 fmol of helicase substrate and, where indicated, 13 pmol of T antigen helicase. Lane 1 (B) and lane 2 (D), substrate heated to 100 C before loading; lane 2 (B) and lane 1 (D), substrate untreated; lanes 3–7, substrate incubated with either no mtDBP (lane 3) or increasing amounts of mtDBP (0.20, 0.60, 1.20 and 3.20 pmol in lanes 4–7, respectively). (E) Displacement analysis in the presence of unbound mtDBP, performed with the substrate lacking the protein binding site. Reactions contained 5 fmol of partial duplex M13mp18 obtained by annealing M13mp18 single-stranded DNA to a complementary 50 end radiolabelled 20mer oligonucleotide: 13 pmol of T antigen and 3.5 pmol of mtDBP were used where indicated. Lane 1, substrate untreated; lane 2, substrate heated to 100 C before loading.