Figure 1. Gel mobility shift analysis of mtDBP to measure the protein:DNA ratio for the helicase assays. (Top) Schematic representation of the non- coding region, NCR, (grey bar) of P.lividus mtDNA displaying the mtDBP binding site occupied by the protein, the downstream G-stretch (G-rich) and the AT-rich sequence (AT). Numbers mark the position on mtDNA of the mtDBP binding site (square bracket) as from DNase I footprinting analysis (9) and the first guanosine of the G-stretch. Some genes flanking the NCR are shown. Horizontal black arrows indicate the direction of RNA transcription; OH refers to the replication origin of the leading DNA strand; open arrow indicates the direction of DNA synthesis. The possible DNA portion of the D-strand is indicated as a bold line, the remaining RNA portion as a thin line. (Bottom) Gel shift analysis with increasing amounts of mtDBP. The assay was performed as described in Materials and Methods, in the presence of 5 fmol of a 46mer double-stranded oligonucleotide, containing the protein binding site, which was 32P-labelled at its 50 ends. NCR is placed in correspondence of the 30 end of the triplex structure, where H-strand DNA synthesis is either pre- maturely terminated or allowed to continue (Figure 1, top panel) (4,11). This observation raised the possibility that mtDBP may be involved in mtDNA replication; in particular, it could act as a negative regulator by antagonizing some enzymes of the replication machinery such as replicative DNA helicases. In the present work, we wished to investigate whether the sea urchin DNA-binding protein mtDBP is able to exert a contrahelicase activity. Since an in vitro replicative system from sea urchin mitochondria is yet to be developed, we have used the helicase activity of the SV40 large T antigen (12), taking advantage of the fact that the viral enzyme has been used in several in vitro heterologous replication systems (13–15), and it has reproduced the properties of the speciﬁc helicases (16,17). The data reported here demonstrate that mtDBP impairs the helicase activity of T antigen when bound to a template that is serving as helicase substrate. The contrahelicase activity is independent of the orientation of the protein bind- ing site with respect to the direction of DNA unwinding; furthermore, the inhibitory effect of mtDBP is increased by the presence of the guanosine-rich sequence ﬂanking mtDBP recognition site. In addition, we show that T7 RNA poly- merase transcribing through DNA–mtDBP complex causes the dissociation of the protein from the complex, thus provid- ing an interesting clue to the mechanism of contrahelicase activity abrogation. Nucleic Acids Research, 2005, Vol. 33, No. 12 3813 MATERIALS AND METHODS Proteins SV40 T antigen, expressed in insect cells and puriﬁed, was a gift of Friedrich Grummt (13). To obtain the recombinant mtDBP, the cDNA coding for the mature version of the protein (9) was ampliﬁed by PCR and inserted into pBAD/His C expression vector (Stratagene) that had been digested with NcoI and EcoRI to eliminate the 6·His tag and the anti- Xpress Epitope. The recombinant protein was expressed in Escherichia coli TOP 10 cells by inducing a log-phase culture with 0.2% L(+)-arabinose for 3 h at 37 C. Bacteria were har- vested by centrifugation and resuspended in 20 ml of lysis buffer (50 mM sodium phosphate buffer, pH 7.8, 400 mM NaCl, 100 mM KCl, 10% glycerol, 0.5% Triton X-100, 2 mM EDTA and 1 mM DTT), containing 1 ml of Protease Inhibitor’s Cocktail (Sigma). The suspension was sonicated on ice in short bursts and centrifuged at 130 000 g for 60 min at +4 C; the supernatant (S130) was diluted to 175 mM NaCl with a buffer containing 50 mM sodium phosphate buffer, pH 7.8, 10% glycerol, 2 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl ﬂuoride and 0.5 mg/ml each of antipain, chymostatin, elastatinal, leupeptin and pepstatin A (Sigma). The diluted S130 was applied to Heparin-Sepharose CL-6B (Amersham Biosciences) equilibrated with a buffer containing 10 mM Tris–HCl, pH 8.0, 150 mM KCl, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM DTT and protease inhibitors, as before. mtDBP was eluted with 500 mM KCl buffer; its buffer was changed into 100 mM KCl by Centricon 10 (Amicon). The purity of the protein was 80% as estimated by SDS–PAGE and Coomassie Brilliant blue staining. Gel mobility shift assay The reactions were performed in a 20 ml volume containing 50 mM Tris–HCl, pH 7.5, 8 mM MgCl2, 0.1 mg/ml BSA, 0.5 mM DTT, 5 fmol of a double-stranded 46mer oligonuc- leotide probe containing the mtDBP binding site that was labelled at its 50-termini with polynucleotide kinase (Roche) and [g -32P]ATP, and varying amounts of mtDBP as indicated in the ﬁgure legend. The reactions were carried out at 25 C for 30 min and resolved on a 6% native polyacrylamide gel in 0.5· Tris-borate–EDTA (TBE) that was dried and analysed by phosphorimaging. Helicase substrates The single-stranded DNAs used in the helicase assays were prepared from recombinant pBluescript II KS(+) plasmids constructed as follows. Plasmid For was made by inserting into BamHI–XhoI digested plasmid a 157 bp fragment [nt 1090–1246, according to Cantatore et al. (6)] prepared by PCR on P.lividus mtDNA. Plasmid Rev was obtained as for plasmid For, except that the ends of the PCR-ampliﬁed DNA fragment were inverted. Single- stranded recombinant pBluescript was rescued by using M13K07 Helper Phage (Invitrogen) according to the manu- facturer’s instructions. Partial duplex DNAs for the heli- case assays were prepared as follows. Oligonucleotides designed on P.lividus mtDNA and consisting of 46 nt (O-DBP.for, nt 1090–1135; O-DBP.rev, nt 1135–1090) and