Nucleic Acids Research, 2005, Vol. 33, No. 12 3817 Figure 3. Effect of the G-rich element on the contrahelicase activity by DNA-bound mtDBP. (A and C) Schematic representation of NCR.For and NCR.Rev helicase substrates used in the unwinding reactions. The 110mer oligonucleotides O-NCR.for and O-NCR.rev, containing the mtDBP binding sequence and the G-stretch (underlined), were end-radiolabelled and annealed to single-stranded DNA to construct the double-stranded region of NCR.For and NCR.Rev substrates, respec- tively. 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 contained 5 fmol of substrate and, where indicated, 13 pmol of T antigen. Lane 1, substrate untreated; lane 2, substrate heated to 100 C before loading; lanes 3–7, substrate incubated with either no mtDBP (lane 3) or increasing amounts of mtDBP (0.02, 0.20, 0.60 and 1.20 pmol in lanes 4–7, respectively). (E) Schematic representation of the partial duplex substrate lacking the G-rich stretch, used in the unwinding reactions. The 110mer oligonucleotide O-NCR-NoGs.for, containing the mtDBP binding sequence (underlined), was 50 end radiolabelled and annealed to the recombinant single-stranded DNA prepared from plasmid pDBP-term2(R) (10). This procedure yielded the partial duplex NCR-NoGs.For substrate. (F) Autoradiogram of the gel showing a representative displacement experiment. Assays contained 5 fmol of substrate and, where indicated, 13 pmol of T antigen. Lane 1, substrate heated to 100 C before loading; lane 2, substrate untreated; lanes 3–7, substrate incubated with either no mtDBP (lane 3) or increasing amounts of mtDBP (0.60, 1.20, 2.4 and 3.2 pmol in lanes 4–7, respectively).