3814 Nucleic Acids Research, 2005, Vol. 33, No. 12 110 nt (O-NCR.for, nt 1090–1199; O-NCR.rev, nt 1199–1090) were labelled with 32P at their 50-termini with polynucleo- tide kinase (Roche) and [g-32P]ATP; a 3-fold excess of each oligonucleotide was annealed to single-stranded DNA in a 10 ml reaction containing 50 mM NaCl, 6.6 mM MgCl2, 6.6 mM Tris–HCl, pH 7.6. This procedure gave rise to substrates DBP.For, DBP.Rev, NCR.For and NCR.Rev. The helicase substrate that does not contain the G-rich stretch in the heteroduplex region (NCR-NoGs.For) was prepared by annealing, as described before, the 110 nt oligonucleotide O-NCR-NoGs.for (see Figure 3E), labelled with 32P at its 50-termini, to single-stranded DNA obtained from the recom- binant plasmid pDBP.term2(R) (10). The unspeciﬁc substrate was prepared by annealing a 50 end-labelled 20mer oligonuc- leotide [nt 6288–6307 of M13mp18(+)] to M13mp18(+) single-stranded DNA (Amersham Biosciences). The annealed DNAs were puriﬁed from unannealed oligonucleotides by Centricon 100 (Amicon) using the annealing buffer, according to the manufacturer’s instructions. DNA helicase assay The reaction mixture (20 ml) contained 50 mM Tris–HCl, pH 7.5, 8 mM MgCl2, 6 mM ATP, 0.1 mg/ml BSA, 50 mM K-Glutamate, 0.5 mM DTT, 13 pmol of SV 40 T antigen, 5 fmol of the DNA substrate and increasing amounts of mtDBP as indicated in ﬁgure legends. mtDBP-containing reactions were preincubated for 30 min at 25 C before adding SV40 T antigen. Reactions were then incubated for 30 min at 37 C and stopped by addition of 2 ml of stop solution (3.3% SDS, 50 mM EDTA, pH 8.0, 30% glycerol, 0.25% bromophenol blue and 0.25% xylene cyanol). The samples with no mtDBP always received the protein buffer corres- ponding to the highest volume of mtDBP used in the assay. Reaction products were separated by electrophoresis through a 10% non-denaturing polyacrylamide gel that was dried and analysed by phosphorimaging. The intensities of the bands were quantiﬁed by densitometry using the program ImageQuant (Molecular Dynamics). Transcription-gel mobility shift assay The template was an 895 bp fragment obtained by PCR on plasmid pDBP-term2(R) (10) using as primers the T7 pro- moter and A.For (nt 490–508) sequences. Template DNA (100 fmol) was incubated with recombinant mtDBP at 25 C for 15 min in a 20 ml mixture containing T7 RNA polymerase buffer [40 mM Tris–HCl, pH 8.0, 25 mM NaCl, 8 mM MgCl2 and 2 mM spermidine-(HCl)3], 28 U of RNaseOUT (Invitro- gen), 0.5 mM each of ATP, CTP and GTP, and 0.05 mM UTP. Five units of T7 RNA polymerase (Invitrogen) were added and incubated for 1 min, following which 12 fmol of a [g-32P]-labelled 46mer double-stranded oligonucleotide containing the mtDBP binding site was added and incuba- tion was continued for 30 min at 32 C. All the reactions, including those in the absence of transcription, contained T7 RNA polymerase buffer and nucleotides unless speciﬁed. The reactions were resolved in a 6% native poly- acrylamide gel in 0.5· TBE that was dried and analysed by phosphorimaging. RESULTS The transcription termination factor mtDBP inhibits the activity of the replicative SV40 helicase in a bidirectional mode In this work, we investigated whether the sea urchin protein mtDBP bound to its cognate site in the NCR of mtDNA is able to exert a contrahelicase activity. To assay this function, we used the replicative helicase of the virus SV40 (large T antigen). This is an ATP-dependent DNA helicase that trans- locates along a single-stranded template in 30 to 50 direction and unwinds DNA hybrids when it encounters a duplex region (12). First, we wished to determine the protein:DNA ratio that yielded a complete saturation of the DNA probe by mtDBP. Therefore, we performed a gel mobility shift assay with dif- ferent amounts of the recombinant protein varying from 0.4 to 5.5 pmol, in the presence of 5 fmol of the labelled probe containing mtDBP binding site. As reported in Figure 1, we observed that, beginning from 2 to 3 pmol up to 5.5 pmol of mtDBP, the DNA was almost completely bound, as very little free DNA was present; therefore we chose 3.2 pmol as the maximum amount of mtDBP for the contrahelicase assays described below. To prepare the speciﬁc helicase substrates, we employed a circular single-stranded recombinant pBluescript bearing, in either orientation, the entire NCR of P.lividus mtDNA that includes mtDBP binding site and its ﬂanking sequence. For the ﬁrst helicase assay, the partial duplex was formed by annealing the 50 end-labelled oligonucleotides containing the mtDBP binding site in both orientations to the comple- mentary sequence present on the single-stranded DNA. This produced two partial duplex substrates, named DBP.For and DBP.Rev, each containing the protein binding site in one possible orientation (Figure 2A and C). Helicase assays were performed by incubating the substrates with the SV40 helicase. As reported in the autoradiograms shown in Figure 2B and D, lane 3, addition of 13 pmol of T antigen resulted, with both substrates, in the release of 60% of the annealed oligonucleotide. When substrate DBP.For was pre- incubated with increasing amounts of recombinant mtDBP and then exposed to the enzyme, we observed a progressive impairment of the helicase activity in a dose-dependent man- ner (panel B, lanes 4–7). When the substrate was reversed, mtDBP inhibited the helicase activity approximately at the same extent (panel D, lanes 4–7). In both cases, the DNA unwinding activity was almost completely abolished by 3.2 pmol of protein. These ﬁndings indicate that mtDBP possesses a bidirectional contrahelicase activity. To rule out the possibility that the T antigen helicase activity could be inhibited in a non-speciﬁc manner by the free mtDBP rather than by the DNA-bound protein, we per- formed a helicase assay with a control non-speciﬁc substrate whose duplex region did not contain mtDBP binding site. No inhibitory effect was observed upon addition to the assay of 3.5 pmol of mtDBP (panel E, lanes 3 and 4). This indicates that the DNA unwinding activity of large T antigen is not inhibited either by the unbound mtDBP or by additional com- ponents present in the mtDBP-containing chromatographic fraction.