Figure 6. Diagram showing the suggested mechanism of abrogation of mtDBP contrahelicase activity by transcriptional invasion of the replication origin. The NCR of P.lividus mtDNA, including mtDBP binding site and the downstream regulatory elements (G-rich and AT-rich regions), is shown. Open arrow indicates the direction of H-strand DNA synthesis; filled arrow refers to the direction of RNA polymerase transcription. See legend to Figure 1 for description. complex causes the dissociation of mtDBP from the DNA. On this basis, we present in Figure 6 a hypothetical model for mtDBP as the device that regulates the interplay between mtDNA transcription and replication. A possible change in the conformational state of bound mtDBP and/or a leakage of transcription termination could allow the movement through the complex of the mitochondrial RNA polymerase that is travelling oppositely to the direction of replication. The tran- scriptional passage would cause the dissociation of mtDBP from its target site and the subsequent abrogation of the helicase impairment. The existence of rare transcripts going beyond the transcription termination site and mapping in the main regulatory region (24) is consistent with the readthrough of the RNA polymerase. Taken together, these data invite speculation that permissive transcription through the DNA– protein complex might provide a mechanism for regulation of mtDNA synthesis. Our data should be a productive avenue for further investigations aimed at shedding light on the interplay between replication and transcription in animal mitochondria. ACKNOWLEDGEMENTS We thank Dr F. Grummt for the gift of SV40 T antigen. The technical assistance of Vito Cataldo is acknowledged. The work was supported by grants from Universita` di Bari (Progetto di Ricerca di Ateneo); Centro di Eccellenza di Genomica in Campo Biomedico e Agrario and Piani di Potenziamento della Rete Scientifica e Tecnologica, Legge 488/92 Cluster C03. S.D. was supported by funds from ESF (PON 2000–2006). Funding to pay the Open Access publica- tion charges for this article was provided by Istituto di Biomembrane e Bioenergetica, CNR, Bari. Conflict of interest statement. None declared. Nucleic Acids Research, 2005, Vol. 33, No. 12 3819 REFERENCES 1. Shadel,G.S. and Clayton,D.A. (1997) Mitochondrial DNA maintenance in vertebrates. Annu. Rev. Biochem., 66, 409–435. 2. Holt,I.J., Lorimer,H.E. and Jacobs,H.T. (2000) Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell, 100, 515–524. 3. Bowmaker,M., Yang,M.Y., Yasukawa,T., Reyes,A., Jacobs,H.T., Huberman,J.A. and Holt,I.J. (2003) Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone. J. Biol. Chem., 278, 50961–50969. 4. Jacobs,H.T., Herbert,E.R. and Rankine,J. (1989) Sea urchin egg mitochondrial DNA contains a short displacement loop (D-loop) in the replication origin region. Nucleic Acids Res., 17, 8949–8965. 5. Jacobs,H.T., Elliott,D.J., Math,V.B. and Farquharson,A. (1988) Nucleotide sequence and gene organization of sea urchin mitochondrial DNA. J. Mol. Biol., 202, 185–217. 6. Cantatore,P., Roberti,M., Rainaldi,G., Gadaleta,M.N. and Saccone,C. (1989) The complete nucleotide sequence, gene organization, and genetic code of the mitochondrial genome of Paracentrotus lividus. J. Biol. Chem., 264, 10965–10975. 7. Madsen,C.S., Ghivizzani,S.C. and Hauswirth,W.W. (1993) Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region. Mol. Cell. Biol., 13, 2162–2171. 8. Roberti,M., Musicco,C., Loguercio Polosa,P., Milella,F., Gadaleta,M.N. and Cantatore,P. (1998) Multiple protein-binding sites in the TAS-region of human and rat mitochondrial DNA. Biochem. Biophys. Res. Commun., 243, 36–40. 9. Loguercio Polosa,P., Roberti,M., Musicco,C., Gadaleta,M.N., Quagliariello,E. and Cantatore,P. (1999) Cloning and characterisation of mtDBP, a DNA-binding protein which binds two distinct regions of sea urchin mitochondrial DNA. Nucleic Acids Res., 27, 1890–1899. 10. Fernandez-Silva,P., Loguercio Polosa,P., Roberti,M., Di Ponzio,B., Gadaleta,M.N., Montoya,J. and Cantatore,P. (2001) Sea urchin mtDBP is a two-faced transcription termination factor with a biased polarity depending on the RNA polymerase. Nucleic Acids Res., 29, 4736–4743. 11. Mayhook,A.G., Rinaldi,A.M. and Jacobs,H.T. (1992) Replication origins and pause sites in sea urchin mitochondrial DNA. Proc. R. Soc. Lond., B, Biol. Sci., 248, 85–94. 12. Stahl,H., Dro¨ge,P. and Knippers,R. (1986) DNA helicase activity of SV40 large tumor antigen. EMBO J., 5, 1939–1944. 13. Putter,V. and Grummt,F. (2002) Transcription termination factor TTF-I exhibits contrahelicase activity during DNA replication. EMBO Rep., 3, 147–152. 14. Bedrosian,C.L. and Bastia,D. (1991) Escherichia coli replication terminator protein impedes simian virus 40 (SV40) DNA replication fork movement and SV40 large tumor antigen helicase activity in vitro at a prokaryotic terminus sequence. Proc. Natl Acad. Sci. USA, 88, 2618–2622. 15. Amin,A.A. and Hurwitz,J. (1992) Polar arrest of the simian virus 40 tumor antigen-mediated replication fork movement in vitro by the tus protein– terB complex of Escherichia coli. J. Biol. Chem., 267, 18612–18622. 16. Lee,E.H., Kornberg,A., Hidaka,M., Kobayashi,T. and Horiuchi,T. (1989) Escherichia coli replication terminator protein impedes the action of helicases. Proc. Natl Acad. Sci. USA, 86, 9104–9108. 17. Sahoo,T., Mohanty,B.K., Lobert,M., Manna,A.C. and Bastia,D. (1995) The contrahelicase activities of the replication terminator proteins of Escherichia coli and Bacillus subtilis are helicase-specific and impede both helicase translocation and authentic DNA unwinding. J. Biol. Chem., 270, 29138–29144. 18. Mohanty,B.K., Sahoo,T. and Bastia,D. (1998) Mechanistic studies on the impact of transcription on sequence-specific termination of DNA replication and vice versa. J. Biol. Chem., 273, 3051–3059. 19. Mulugu,S., Potnis,A., Shamsuzzaman, Taylor,J., Alexander,K. and Bastia,D. (2001) Mechanism of termination of DNA replication of Escherichia coli involves helicase–contrahelicase interaction. Proc. Natl Acad. Sci. USA, 98, 9569–9574. 20. Mohanty,B.K., Sahoo,T. and Bastia,D. (1996) The relationship between sequence-specific termination of DNA replication and transcription. EMBO J., 15, 2530–2539. 21. Mohanty,B.K., Bussiere,D.E., Sahoo,T., Pai,K.S., Meijer,W.J., Bron,S. and Bastia,D. (2001) Structural and functional analysis of a bipolar replication terminus. Implications for the origin of polarity of fork arrest. J. Biol. Chem., 276, 13160–13168.