3812–3820 Nucleic Acids Research, 2005, Vol. 33, No. 12 doi:10.1093/nar/gki693 Contrahelicase activity of the mitochondrial transcription termination factor mtDBP Paola Loguercio Polosa 1 , Stefania Deceglie1, Marina Roberti1, Maria Nicola Gadaleta1,2 and Palmiro Cantatore1,2,* 1Dipartimento di Biochimica e Biologia Molecolare, Universita` degli Studi di Bari, Via Orabona, 4, 70125 Bari, Italy and 2Istituto di Biomembrane e Bioenergetica, CNR, Via Amendola, 165/A, 70126 Bari, Italy Received March 15, 2005; Revised June 3, 2005; Accepted June 16, 2005 ABSTRACT The sea urchin mitochondrial D-loop binding protein (mtDBP) is a transcription termination factor that is able to arrest bidirectionally mitochondrial RNA chain elongation. The observation that the mtDBP binding site in the main non-coding region is located in correspondence of the 30 end of the triplex struc- ture, where the synthesis of heavy strand mito- chondrial (mt) DNA is either prematurely terminated or allowed to continue, raised the question whether mtDBP could also regulate mtDNA replication. By using a helicase assay in the presence of the replic- ative helicase of SV40, we show that mtDBP is able to inhibit the enzyme thus acting as a contraheli- case. The impairing activity of mtDBP is bidirectional as it is independent of the orientation of the protein binding site. The inhibition is increased by the pres- ence of the guanosine-rich sequence that flanks mtDBP binding site. Finally, a mechanism of abroga- tion of mtDBP contrahelicase activity is suggested that is based on the dissociation of mtDBP from DNA caused by the passage of the RNA polymerase through the protein–DNA complex. All these findings favour the view that mtDBP, besides serving as transcription termination factor, could also act as a negative regulator of mtDNA synthesis at the level of D-loop expansion. INTRODUCTION According to the traditional model, mammalian mitochondrial (mt) DNA replicates in a continuous, unidirectional and strand-asymmetric mode, starting from two speciﬁc origins of replication, OH for the heavy (H) or leading strand, and OL for the light (L) or lagging strand (1). Only a few initiation events at OH result in the production of a complete genome; the nascent H-strand frequently terminates 500–1000 bp downstream of OH (depending on the species) and, since the terminated strand remains annealed to the template, a typical triple-stranded structure is created that is called the displace- ment (D) loop. Recently, an additional replication model of coupled leading and lagging-strand mtDNA synthesis has been proposed (2,3). In sea urchin, the present knowledge on mtDNA replication relies on early studies by Jacobs and coworkers who suggested the presence of an OH in the main non-coding region (NCR) of Strongylocentrotus purpuratus mtDNA (4). The NCR ( 130 bp long) is much shorter than in vertebrates and includes a string of continuous guanosines and an AT-rich sequence that probably functions as transcriptional promoter. The D-loop structure associated with OH is very short ( 80 bp) and the nascent strand seems to consist of an RNA primer of 60 nt joined to a small stretch of DNA chain (Figure 1, top panel), (5,6). Regarding the molecular mechanisms that control the expansion of the D-loop structure, some DNA-binding activ- ities with a putative role in this event have been identiﬁed in several species. In bovine Madsen et al. (7) characterized a trans-acting factor of 48 kDa, which binds a conserved sequence element (TAS, termination associated sequence) located upstream of the D-loop 30 end. By means of in vivo footprinting, we detected in rat and human mitochondria two protein binding sites in the 30 region of the D-loop (8). In the Mediterranean species of sea urchin Paracentrotus lividus we identiﬁed the mitochondrial DNA-binding protein mtDBP (9). It binds two regions on sea urchin mtDNA, one located in the NCR, the other placed at the boundary of the oppositely transcribed genes for ND5 and ND6. Recently, we showed that mtDBP serves as bidirectional transcription termination factor, able to arrest RNA chain elongation on both strands (10). Interestingly, mtDBP binding site in the *To whom correspondence should be addressed at Dipartimento di Biochimica e Biologia Molecolare, Universita` degli Studi di Bari, Via Orabona, 4, 70125 Bari, Italy. Tel: +39 080 5443378; Fax: +39 080 5443403; Email: p.cantatore@biologia.uniba.it The Author 2005. Published by Oxford University Press. All rights reserved. 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