3816 Nucleic Acids Research, 2005, Vol. 33, No. 12 Having established that mtDBP possesses a contrahelicase activity, we asked whether such capacity was affected by the sequence ﬂanking the protein binding site, in particular the 25-guanosine rich sequence that is placed 50 bp down- stream of mtDBP target site (Figure 1, top panel). To address this point, we constructed two partial duplex substrates (NCR.For and NCR.Rev) by annealing the single-stranded recombinant pBluescript to two 50 end-labelled oligonuc- leotides spanning from mtDBP binding site to the stretch of guanosine residues, with both orientations (Figure 3A and C). The results of the experiments are reported in panels B and D. In the absence of mtDBP, the helicase caused DNA unwinding that released 50% of the annealed oligonucleo- tide with both substrates (panels B and D: lane 3). Since the extent of release was similar to that observed with the short oligonucleotides, it results that neither the length of the heteroduplex region nor the presence of the G-rich element affected substantially the helicase activity. Addition of increasing amounts of mtDBP caused, with both substrates NCR.For and NCR.Rev, a progressive impairment of the hel- icase activity that was almost completely abolished already by 1.2 pmol of protein in the assay (panels B and D, lanes 4–7). The inhibitory effect of mtDBP did not remarkably change with the orientation of the binding site, thus conﬁrming that mtDBP contrahelicase activity is orientation-independent. At this point, we endeavoured to test whether such increased helicase inhibition by mtDBP was caused by the G-rich ele- ment or rather by the fact that the heteroduplex region was longer (110 bp as compared with 46 bp). We constructed a helicase substrate lacking the G-string, whose partial duplex region consisted of a 75 bp region of sea urchin mtDNA bearing the mtDBP binding site in the forward orientation, ﬂanked by additional sequences so as to yield a double- stranded region of 110 bp (Figure 3E). The autoradiogram displayed in panel F shows that the extent of helicase arrest by mtDBP on this substrate was substantially similar to that observed with the 46 bp duplex substrate (Figure 2B and D). A similar result was obtained with the substrate in the opposite orientation (data not shown). These data indicate that the ability of mtDBP to impede T antigen helicase activity was not substantially affected by the length of the hetero- duplex region in the substrate but, rather, by the presence of the uninterrupted 25 bp guanosine sequence. Figure 4 sum- marizes the results of the helicase assays. It shows that, with the two long substrates containing mtDBP binding site and the G-rich stretch (NCR.For and NCR.Rev), 50% inhibition of the helicase activity was achieved with 0.3 pmol of mtDBP. Almost a 4-fold excess of mtDBP was however needed to reach the same level of helicase impairment on the short sub- strates DBP.For and DBP.Rev as well as on the long hetero- duplex substrate lacking the G-rich stretch (NCR-NoGs.For, top panel). Thus, we conclude that the presence of a G-rich element in the substrate reinforces the contrahelicase effect of mtDBP. The DNA-bound mtDBP is dislodged by an invading RNA transcript It has been demonstrated that in bacteria, when T7 RNA poly- merase transcribes through the complex formed by the con- trahelicase Tus and its cognate site on DNA, dislodging of the protein from DNA occurs, that is followed by abrogation of helicase impediment (18). As a ﬁrst step towards investigating whether a similar mechanism may take place also in sea urchin mitochondria, we analysed the capability of T7 RNA poly- merase to dislodge bound mtDBP from its cognate DNA site when the enzyme is allowed to pass through the DNA–mtDBP complex. T7 RNA polymerase was chosen since we had pre- viously shown that the enzyme is not arrested by the DNA- bound mtDBP when it travels in the direction opposite to that of DNA replication (10). We performed an experiment whose strategy is depicted in Figure 5, top panel. Brieﬂy, a transcription assay was carried out on a template contain- ing the T7 promoter upstream of the mtDBP binding site. A 32P-labelled, double-stranded 46mer oligonucleotide con- taining the mtDBP site was added to the mixture to trap the protein that might be dislodged from the template during tran- scription. The values of DNA template–protein ratios used in the assay were such that there was undetectable or little free protein available for gel shift. The results of the experiment are shown in Figure 5, bottom panel. Whereas in the absence of transcription, due to the lack of RNA polymerase or NTPs in the assay, very little mobility shift was obtained (lanes 1, 3 and 4), transcription of the template produced a retarded band whose intensity was much stronger (compare lane 2 with 1 and lane 5 with 4). The retarded band had the same size as that produced by the puriﬁed mtDBP (compare lanes 2 and 5 with lane 7). These results can be explained by assuming that the passage of T7 RNA polymerase through the DNA–mtDBP complex caused the dissociation of mtDBP that was trapped by the labelled probe. DISCUSSION We have previously inferred that the DNA-binding protein mtDBP could play a complex role in regulating sea urchin mtDNA expression (10). The ability of mtDBP to bidirec- tionally arrest the progression of mtRNA polymerase would mainly serve two functions: to generate the 30 end of H- and L-strand transcripts and, at the same time, to control the pas- sage of the transcribing enzymes that are moving in opposite directions. Furthermore, since the sea urchin main NCR is a condensed version of the vertebrate regulatory region, with transcriptional and replicative elements being co-localized in a much shorter space (4), we proposed that mtDBP could also participate in regulating the mtDNA replicative process (10). In particular, mtDBP, bound to the target sequence placed near the 30 end of the D-loop structure, could counteract enzyme(s) of the replicative machinery and determine the formation of the triple-stranded structure, thus acting as negative regulator of mtDNA replication. The results presented in this paper strongly support this view. We show that the mitochondrial transcription termination factor mtDBP possesses a contrahel- icase activity. In fact, using partial duplex substrates that contain the protein binding site and its ﬂanking region, we ﬁnd that DNA-bound mtDBP inhibits bidirectionally the helicase activity of SV40 large T antigen. The sea urchin factor is to our knowledge the ﬁrst documented example of a mito- chondrial DNA-binding protein that, besides arresting RNA transcription, is also able to impede the DNA unwinding activity of a replicative helicase.