3818 Nucleic Acids Research, 2005, Vol. 33, No. 12 Figure 4. Quantitative analyses of the displacement reactions. Representative standard curves of the percentage of DNA unwinding by SV40 T antigen on substrates DBP.For, NCR.For and NCR-NoGs.For (Top) and DBP.Rev, NCR.Rev (Bottom) are shown. The percentage of DNA unwinding was plotted against the amount of mtDBP; dotted lines indicate the amount of protein needed to obtain 50% inhibition of the helicase activity. Extent of release of labelled oligonucleotides in the absence of mtDBP was set as 100%. Proteins acting as transcription terminators and displaying a contrahelicase activity have been described in prokaryotic as well as in eukaryotic systems. The replication termination factor Tus in E.coli binds to its cognate terminator sequence and arrests the replication fork in a polar manner by estab- lishing speciﬁc protein–protein contacts with the replicative helicase (19). The same factor is also able to exert a polar block to RNA chain elongation (20). In eukaryotes, the DNA- bound transcription terminator factor TTF-I, which promotes arrest of RNA polymerase I, was also shown to terminate replication of the rDNA replicon by impeding in a polar manner the helicase activity of SV40 large T antigen (13). Interestingly, we found that mtDBP stops the T antigen helicase independently on the orientation of its binding site. The bipolar functionality of mtDBP agrees with its ability to arrest bidirectionally the elongating mitochondrial RNA polymerase. Similarly, the prokaryotic replication terminator protein RTP, when bound to the bipolar replication terminus of the plasmid pLS20, was shown to arrest both DNA helicase and RNA polymerase in a bidirectional mode (21). This evidence indicates that the polarity of transcriptional arrest is generally consistent with that of helicase impairment. The bidirectionality of mtDBP contrahelicase activity might be used to block helicases that unwind DNA from both directions, as it would occur if mtDNA replicates by the bidirectional strand-coupled mechanism (3). The presence of the uninterrupted 25-guanosine stretch downstream of the mtDBP binding site reinforces the inhib- itory effect of the protein, since such enhancement is not observed when a substrate of the same length but lacking the G-string is used in the contrahelicase assay. It is well Figure 5. Fate of mtDBP when T7 RNA polymerase transcribes through mtDBP–DNA complex, as determined by combining RNA transcription with gel mobility shift assays. (Top) Diagrammatic representation of the experimental strategy. (Bottom) Autoradiogram of a 6% polyacrylamide gel showing the results of gel shift assay with the 32P-labelled 46mer. Reac- tions were performed as described in Materials and Methods. DNA template: protein ratios were 1:6 and 1:13 in lanes 1–3 and 4–7, respectively. A trans- cription assay in the presence of [a-32P]UTP was performed in parallel (data not shown). known that G-rich sequences can form higher order structures, such as a triple helix consisting of poly (dG-dG-dC) strands (22,23). However, the G-string doesn’t seem to possess an intrinsic contrahelicase activity since we found that the annealed oligonucleotides containing such element are extens- ively released by the viral helicase. Thus, our ﬁndings point to a functional cooperativity between the DNA-bound protein and its ﬂanking region in determining the arrest of the helicase. The role of G-rich elements in mediating the contrahelicase activity of a replication terminator has been reported also for TTF-I; in this case, the G-stretch is indispensable for the protein to exert its inhibitory activity (13). The capability of mtDBP to arrest both the replicative hel- icase and the elongating mtRNA polymerase suggests that mtDNA replication and transcription are mutually interacting in the main NCR of sea urchin mitochondria. Some clues to a possible mechanism could be surmised from the mechanism of replication termination in prokaryotes. It has been reported that transcription invasion of the replication terminus abrog- ates the contrahelicase activity of Tus, as a result of its dis- location from DNA (18). We have investigated the impact of transcription on the DNA-bound mtDBP and have noted that the passage of the RNA polymerase through the protein–DNA