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Polosa PL
,
Deceglie S
,
Roberti M
,
Gadaleta MN
,
Cantatore P
.
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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 3'' end of the triplex structure, where the synthesis of heavy strand mitochondrial (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 replicative helicase of SV40, we show that mtDBP is able to inhibit the enzyme thus acting as a contrahelicase. 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 presence of the guanosine-rich sequence that flanks mtDBP binding site. Finally, a mechanism of abrogation 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.
Figure 1. Gel mobility shift analysis of mtDBP to measure the protein:DNA ratio for the helicase assays. (Top) Schematic representation of the non-coding region, NCR, (grey bar) of P.lividus mtDNA displaying the mtDBP binding site occupied by the protein, the downstream G-stretch (G-rich) and the AT-rich sequence (AT). Numbers mark the position on mtDNA of the mtDBP binding site (square bracket) as from DNase I footprinting analysis (9) and the first guanosine of the G-stretch. Some genes flanking the NCR are shown. Horizontal black arrows indicate the direction of RNA transcription; OH refers to the replication origin of the leading DNA strand; open arrow indicates the direction of DNA synthesis. The possible DNA portion of the D-strand is indicated as a bold line, the remaining RNA portion as a thin line. (Bottom) Gel shift analysis with increasing amounts of mtDBP. The assay was performed as described in Materials and Methods, in the presence of 5 fmol of a 46mer double-stranded oligonucleotide, containing the protein binding site, which was 32P-labelled at its 5′ ends.
Figure 2. Inhibition of SV40 T antigen helicase by DNA-bound mtDBP. (A and C) Schematic representation of DBP.For and DBP.Rev partial duplex substrates used in the unwinding reactions. The 46mer oligonucleotides O-DBP.for and O-DBP.rev containing the mtDBP binding sequence (underlined) were end-radiolabelled and annealed to the recombinant single-stranded DNA to construct the double-stranded region of DBP.For and DBP.Rev substrates, respectively. Numbers give the position of sea urchin mtDNA nucleotides; open arrow marks the orientation of the protein target site with respect to T antigen unwinding direction (black arrow). (B and D) Autoradiograms of representative displacement analyses showing the annealed partial duplex substrate (upper band) and the released oligonucleotide (lower band). Assays were performed as described in Materials and Methods, in the presence of 5 fmol of helicase substrate and, where indicated, 13 pmol of T antigen helicase. Lane 1 (B) and lane 2 (D), substrate heated to 100°C before loading; lane 2 (B) and lane 1 (D), substrate untreated; lanes 3–7, substrate incubated with either no mtDBP (lane 3) or increasing amounts of mtDBP (0.20, 0.60, 1.20 and 3.20 pmol in lanes 4–7, respectively). (E) Displacement analysis in the presence of unbound mtDBP, performed with the substrate lacking the protein binding site. Reactions contained 5 fmol of partial duplex M13mp18 obtained by annealing M13mp18 single-stranded DNA to a complementary 5′ end radiolabelled 20mer oligonucleotide: 13 pmol of T antigen and 3.5 pmol of mtDBP were used where indicated. Lane 1, substrate untreated; lane 2, substrate heated to 100°C before loading.
Figure 3. Effect of the G-rich element on the contrahelicase activity by DNA-bound mtDBP. (A and C) Schematic representation of NCR.For and NCR.Rev helicase substrates used in the unwinding reactions. The 110mer oligonucleotides O-NCR.for and O-NCR.rev, containing the mtDBP binding sequence and the G-stretch (underlined), were end-radiolabelled and annealed to single-stranded DNA to construct the double-stranded region of NCR.For and NCR.Rev substrates, respectively. Numbers give the position of sea urchin mtDNA nucleotides; open arrow marks the orientation of the protein target site with respect to T antigen unwinding direction (black arrow). (B and D) Autoradiograms of representative displacement analyses showing the annealed partial duplex substrate (upper band) and the released oligonucleotide (lower band). Assays contained 5 fmol of substrate and, where indicated, 13 pmol of T antigen. Lane 1, substrate untreated; lane 2, substrate heated to 100°C before loading; lanes 3–7, substrate incubated with either no mtDBP (lane 3) or increasing amounts of mtDBP (0.02, 0.20, 0.60 and 1.20 pmol in lanes 4–7, respectively). (E) Schematic representation of the partial duplex substrate lacking the G-rich stretch, used in the unwinding reactions. The 110mer oligonucleotide O-NCR-NoGs.for, containing the mtDBP binding sequence (underlined), was 5′ end radiolabelled and annealed to the recombinant single-stranded DNA prepared from plasmid pDBP-term2(R) (10). This procedure yielded the partial duplex NCR-NoGs.For substrate. (F) Autoradiogram of the gel showing a representative displacement experiment. Assays contained 5 fmol of substrate and, where indicated, 13 pmol of T antigen. Lane 1, substrate heated to 100°C before loading; lane 2, substrate untreated; lanes 3–7, substrate incubated with either no mtDBP (lane 3) or increasing amounts of mtDBP (0.60, 1.20, 2.4 and 3.2 pmol in lanes 4–7, respectively).
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%.
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. Reactions 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 transcription assay in the presence of [α-32P]UTP was performed in parallel (data not shown).
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.
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