Bidirectional Replication Initiates at Sites Throughout the Mitochondrial Genome of Birds

Analysis of mitochondrial replication intermediates of Gallus gallus on fork-direction gels indicates that replication occurs in both directions around circular mitochondrial DNA. This finding was corroborated by a study of chick mitochondrial DNA on standard neutral two-dimensional agarose gels, which yielded archetypal initiation arcs in fragments covering the entire genome. There was, however, considerable variation in initiation arc intensity. The majority of initiation events map to regions flanking the major non-coding region, in particular the NADH dehydrogenase subunit 6 (ND6) gene. Initiation point mapping of the ND6 gene identified prominent free 5′ ends of DNA, which are candidate start sites for DNA synthesis. Therefore we propose that the initiation zone of G. gallus mitochondrial DNA encompasses most, if not all, of the genome, with preferred initiation sites in regions flanking the major non-coding region. Comparison with mammals suggests a common mechanism of initiation of mitochondrial DNA replication in higher vertebrates. Analysis of mitochondrial replication intermediates of Gallus gallus on fork-direction gels indicates that replication occurs in both directions around circular mitochondrial DNA. This finding was corroborated by a study of chick mitochondrial DNA on standard neutral two-dimensional agarose gels, which yielded archetypal initiation arcs in fragments covering the entire genome. There was, however, considerable variation in initiation arc intensity. The majority of initiation events map to regions flanking the major non-coding region, in particular the NADH dehydrogenase subunit 6 (ND6) gene. Initiation point mapping of the ND6 gene identified prominent free 5′ ends of DNA, which are candidate start sites for DNA synthesis. Therefore we propose that the initiation zone of G. gallus mitochondrial DNA encompasses most, if not all, of the genome, with preferred initiation sites in regions flanking the major non-coding region. Comparison with mammals suggests a common mechanism of initiation of mitochondrial DNA replication in higher vertebrates. The study of initiation of θ replication in prokaryotes, viruses, and plasmids led to the discovery of a single discrete origin for each genome, which was essential for replication (1Kornberg A. Baker T.A. DNA Replication. W. H. Freeman & Co., NY1992: 51-52Google Scholar). The large chromosome size of nuclear DNA necessitates multiple origins of replication (2Huberman J.A. Riggs A.D. J. Mol. Biol. 1968; 32: 327-341Crossref PubMed Scopus (631) Google Scholar); nevertheless, it was widely assumed that nuclear DNA would initiate replication at a discrete site for a given region of DNA. The identification of autonomously replicating sequence elements seemed to support this idea (3Van Houten J.V. Newlon C.S. Mol. Cell. Biol. 1990; 10: 3917-3925Crossref PubMed Scopus (138) Google Scholar). However, nuclear DNA replication often initiates at a multiplicity of sites across a broad zone (4Zhou J. Ashouian N. Delepine M. Matsuda F. Chevillard C. Riblet R. Schildkraut C.L. Birshtein B.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13693-13698Crossref PubMed Scopus (44) Google Scholar, 5Little R.D. Platt T.H. Schildkraut C.L. Mol. Cell. Biol. 1993; 13: 6600-6613Crossref PubMed Scopus (207) Google Scholar, 6Vaughn J.P. Dijkwel P.A. Hamlin J.L. Cell. 1990; 61: 1075-1087Abstract Full Text PDF PubMed Scopus (195) Google Scholar, 7Aladjem M.I. Rodewald L.W. Lin C.M. Bowman S. Cimbora D.M. Brody L.L. Epner E.M. Groudine M. Wahl G.M. Mol. Cell. Biol. 2002; 22: 442-452Crossref PubMed Scopus (50) Google Scholar). Even the EBV genome, which contains a site that behaves like a classic discrete origin when cut out of the genome and placed in a plasmid, is replicated from numerous origins distributed over a broad zone when it is intact (8Little R.D. Schildkraut C.L. Mol. Cell. Biol. 1995; 15: 2893-2903Crossref PubMed Scopus (71) Google Scholar). Nor is the initiation zone size-fixed; in flies, the initiation zone size is dependent on developmental stage (9Lunyak V.V. Ezrokhi M. Smith H.S. Gerbi S.A. Mol. Cell. Biol. 2002; 22: 8426-8437Crossref PubMed Scopus (49) Google Scholar). Recently, we reported that the 16.5-kb circles of mammalian mitochondrial DNA (mtDNA) initiate replication from multiple sites across a zone of ∼4 kilobases (10Bowmaker M. Yang M.Y. Yasukawa T. Reyes A. Jacobs H.T. Huberman J.A. Holt I.J. J. Biol. Chem. 2003; 278: 50961-50969Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Although there is great diversity in the size and organization of mitochondrial genomes of plants, fungi, and animals (11Gray M.W. Burger G. Lang B.F. Science. 1999; 283: 1476-1481Crossref PubMed Scopus (1336) Google Scholar), within the animal kingdom they are very similar (12Saccone C. De Giorgi C. Gissi C. Pesole G. Reyes A. Gene (Amst.). 1999; 238: 195-209Crossref PubMed Scopus (348) Google Scholar), particularly so among vertebrates (13Boore J.L. Nucleic Acids Res. 1999; 27: 1767-1780Crossref PubMed Scopus (2361) Google Scholar). One might therefore anticipate a conserved mechanism of replication for vertebrate mtDNA. In the 1970s and 80s, a series of studies of mammalian mtDNA gave rise to a strand-asynchronous model of mtDNA replication (14Clayton D.A. Cell. 1982; 28: 693-705Abstract Full Text PDF PubMed Scopus (913) Google Scholar). The model proposed that replication of the two strands of the circle arose in each case from a single initiation site. These sites were designated the heavy and light strand origins of replication (OH 1The abbreviations used are: OH, heavy strand origin of replication; OL, light strand origin of replication; NCR, non-coding region; np, nucleotide pairs; AGE, agarose gel electrophoresis; LM-PCR, ligation-mediated PCR; ND6, NADH dehydrogenase subunit 6 gene. 1The abbreviations used are: OH, heavy strand origin of replication; OL, light strand origin of replication; NCR, non-coding region; np, nucleotide pairs; AGE, agarose gel electrophoresis; LM-PCR, ligation-mediated PCR; ND6, NADH dehydrogenase subunit 6 gene. and OL). The site of second-strand synthesis, OL, is a sequence element that can theoretically form a hairpin stem-loop and to which abundant free 5′ ends map. Its primary sequence is poorly conserved in mammals, yet the ability to form a stem-loop is conserved (15Hixson J.E. Wong T.W. Clayton D.A. J. Biol. Chem. 1986; 261: 2384-2390Abstract Full Text PDF PubMed Google Scholar). In its simplest or most literal form, this model cannot apply to all vertebrates. Avian mtDNAs lack a convincing stem-loop structure that might act as a dedicated initiation site for second-strand synthesis, begging the question by what mechanism is avian mtDNA replicated? Birds do share with mammals the abundant short displacement or D-loop form of mtDNA (16Kasamatsu H. Robberson D.L. Vinograd J. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 2252-2257Crossref PubMed Scopus (217) Google Scholar, 17Nass M.M. Curr. Genet. 1995; 28: 401-409Crossref PubMed Scopus (15) Google Scholar), which until recently was widely regarded as supporting the strand-asynchronous model. As in mammals, the 3′ end of the D-loop is close to one end of the major non-coding region (NCR), and the 5′ end of the D-loop defines OH. At 16,775 nucleotide pairs (np), the mitochondrial genome of Gallus gallus is slightly larger than that of most mammals (18Desjardins P. Morais R. J. Mol. Biol. 1990; 212: 599-634Crossref PubMed Scopus (855) Google Scholar). Avian D-loops, which typically span almost 800 nucleotides (np 75–855 in G. gallus), account for much of the length difference. The high degree of similarity in gene content and arrangement between avian and mammalian mtDNA is illustrated in Fig. 1.Fig. 3Replication forks travel in both directions in avian mtDNA, yet most forks travel away from the 3′ end of the D-loop. Both potential Y arcs were detectable in fragments of chick mtDNA from around the genome analyzed on fork-direction gels (A, D, G, and J) interpreted in B, E, H, and K. C, F, I, and L, standard single digestion Brewer-Fangman gels. Generally the most prominent arc was associated with replication forks traveling away from the 3′ end of the D-loop, as denoted by 3′ above the arc; a notable exception was the MboI fragment spanning np 14,866–16,476, where in-gel digestion with HincII gave rise to arcs of roughly equal prominence (J). Restriction enzymes are indicated on each panel, or the adjacent panel. Restriction fragments are depicted as lines beneath the related gel images, the enzymes applied, restriction sites, and fragment lengths are marked, together with the position of the probe (filled black box).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2G. gallus mtDNA replication initiates across a region of several kilobases downstream of the 3′ end of the D-loop. Gradient-purified chick liver mtDNA was digested with a series of restriction enzymes, and the products were separated by two-dimensional AGE. A 7.8-kb AflIII fragment (np 10,540–1,603) revealed by hybridization with probe 1, without (A) and with S1 nuclease treatment (B). C, probe 1 was also used to reveal a 4-kb DraI fragment (np 14,075–988) containing OH (np 855) near one end. Cleavage with BsmBI yielded a fragment (np 10,092–768) with one end close to but not including OH (D). The samples in E and F were digested and hybridized with probes that detected, respectively, a FauI fragment np 8,831–16,351, and an MscI fragment np 8,334–15,947. G, the fragments analyzed in A–F are shown aligned with the relevant section of the G. gallus mitochondrial genome (np 7,000–1,000). The D-loop region is depicted as a broad line whose 5′ end is OH, the intensity of each initiation arc is indicated by a number of "+" symbols to the right of each restriction fragment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 1Organization of the mammalian and avian mitochondrial genomes. The gene content of mammalian (A) and avian (B) mitochondrial genomes is the same. Each encodes 13 polypeptides and the RNA elements necessary for their translation. ND, NADH dehydrogenase; CYTB, cytochrome b; CO, cytochrome c oxidase; rRNA, ribosomal RNA genes. The 22 transfer RNAs are denoted according to the single letter amino acid code. The D-loop region of G. gallus is 5′–3′ np 855–75 (18Desjardins P. Morais R. J. Mol. Biol. 1990; 212: 599-634Crossref PubMed Scopus (855) Google Scholar). The arrangement of tRNAGlu and ND6 genes means that tRNAGlu (E) marks the end of the NCR and the 3′ end of the D-loop in birds, whereas these elements abut the tRNAPro gene in mammalian mtDNA. The only other notable difference in structure is the absence of a putative stem-loop structure (OL) in the 5 tRNA gene cluster (YCNAW) of birds. C, schematic map of chick mtDNA, indicating the restriction sites related to the fragments analyzed by two-dimensional AGE in Figs. 2 and 4. The position of the NCR (np 1–1227) is marked as a broad, solid gray line on the outside of the circle. Regional probes 1–5 are depicted as broad, solid black lines on the inside of the circle.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The strand-asynchronous model of mammalian mtDNA replication has been challenged in recent years (19Holt I.J. Lorimer H.E. Jacobs H.T. Cell. 2000; 100: 515-524Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 20Yang M.Y. Bowmaker M. Reyes A. Vergani L. Angeli P. Gringeri E. Jacobs H.T. Holt I.J. Cell. 2002; 111: 495-505Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Duplex replication intermediates were identified that could not be reconciled with the strand-asynchronous model (19Holt I.J. Lorimer H.E. Jacobs H.T. Cell. 2000; 100: 515-524Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar), and the co-existing population of partially single-stranded molecules was subsequently shown to arise from RNase H degradation occurring during isolation of replication intermediates, which contain extensive tracts of RNA-DNA hybrid (20Yang M.Y. Bowmaker M. Reyes A. Vergani L. Angeli P. Gringeri E. Jacobs H.T. Holt I.J. Cell. 2002; 111: 495-505Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Extensive ribonucleotide incorporation has not been reported previously, and it was not known whether this feature was unique to mammalian mtDNA. The contentious nature of recent findings in mammals (21Bogenhagen D.F. Clayton D.A. Trends Biochem. Sci. 2003; 28: 357-360Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 22Holt I.J. Jacobs H.T. Trends Biochem. Sci. 2003; 28: 355-356Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), coupled with the dearth of information on mtDNA replication mechanisms in other vertebrates prompted us to investigate replication intermediates derived from purified mitochondria of the avian G. gallus. The results indicate that bidirectional replication occurs at sites dispersed throughout the mitochondrial genome of G. gallus giving rise to replication forks that travel in both directions around the circle of mtDNA. However, initiation of replication is not random, as most initiation events map to a region adjacent to the 3′ end of the D-loop, centered on the NADH dehydrogenase 6 gene. Purification of Chicken Liver Mitochondria—Chicken liver mitochondria were isolated first by a crude differential centrifugation procedure and subsequently on a single step sucrose density gradient. 20–30 chick livers were minced finely with scissors and washed three or more times with 100 ml of homogenization buffer (HB) comprising 225 mm mannitol, 75 mm sucrose, 10 mm Tris-HCl, pH 7.6, 1 mm EDTA, and 0.1% bovine serum albumin (fatty acid-free). After washing the chopped liver was suspended in 10 ml/g of wet weight of HB buffer and subjected to six strokes of Dounce homogenization with a tight fitting pestle. All operations were performed at 4 °C or on ice. The homogenate was centrifuged at 1,000 × gmax for 5 min, and the supernatant recentrifuged at 9,000 × gmax for 10 min. The crude mitochondrial pellet was resuspended in 30–50 ml HB and loaded onto a sucrose gradient (1.0/1.5 m) and centrifuged at 40,000 × gmax for 120 min. Mitochondria were removed from the interface, diluted in HB, and pelleted by centrifugation at 12,000 × gmax for 15 min. Purified mitochondria were resuspended in 75 mm NaCl, 50 mm EDTA, pH 8.0, 1% SDS, 0.5 mg/ml proteinase K and incubated either for 2 h at 50 °C. Nucleic acid was extracted from the protease-treated mitochondrial pellet by successive incubations with phenol and chloroform, ethanol precipitated, washed and dried, resuspended in 10 mm Tris-HCl, 1 mm EDTA, pH 8.0, and stored at –20 °C. Mouse mtDNA was isolated by the same method (20Yang M.Y. Bowmaker M. Reyes A. Vergani L. Angeli P. Gringeri E. Jacobs H.T. Holt I.J. Cell. 2002; 111: 495-505Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). DNA Digestions—DNA prepared from mitochondria was digested with one of a number of restriction endonucleases under conditions recommended by the manufacturer (New England Biolabs). Where indicated RNase H (Promega) treatment was 1 unit of enzyme for 1 h at 37 °C with 0.1–1.2 μg of mtDNA. RNase One (Promega) treatment was 5 units of enzyme for 10 min at 37 °C with 0.1–1.2 μg of mtDNA. S1 nuclease (Promega) treatment was 1 unit for 1.5 min at 37 °C. For RNase H digestion samples were incubated in 20 mm HEPES-KOH, pH 7.8, 50 mm KCl, 10 mm MgCl2, 1 mm dithiothreitol. RNase One and S1 nuclease treatments employed buffers supplied by the manufacturer (Promega). Two-dimensional Agarose Gel Electrophoresis and Hybridization— Neutral/neutral two-dimensional agarose gel electrophoresis (AGE) was performed by the standard method (23Friedman K.L. Brewer B.J. Methods Enzymol. 1995; 262: 613-627Crossref PubMed Scopus (204) Google Scholar). For fragments of 3–4 kbp, first dimension electrophoresis was 0.7 V/cm for 20 h at room temperature in a 0.4% agarose gel, and second dimension electrophoresis was 6 V/cm for 4 h at 4 °C through a 1% agarose gel, with 300 ng/ml ethidium bromide. In the case of fragments of >5 kb, first dimension electrophoresis was in a 0.35% agarose gel at 1.5 V/cm for 20 h, and second dimension electrophoresis was at 3 V/cm for 18 h in a 0.875% agarose gel. In-gel digestion for fork-direction gels was carried out as follows, after separation of DNA in the first dimension, the gel lane was excised and washed twice with 10 mm Tris, 0.1 mm EDTA, pH 8.0, for 30 min at room temperature. The gel slice was twice equilibrated with 1× restriction enzyme buffer (New England Biolabs) for 1 h at room temperature. Excess buffer was aspirated, and 100 units of restriction enzyme were added directly to the surface of the gel. After incubation at 37 °C for 3 h, a further 50 units of enzyme were added, and the incubation was continued overnight. After overnight incubation, the gel slice was washed first with 10 mm Tris, 1 mm EDTA, pH 8.0, for 30 min at room temperature and then with Tris borate EDTA 1× for 15 min at room temperature. Thereafter, the procedure was identical to standard second dimension electrophoresis for two-dimensional AGE. After Southern blotting, specific regions of chick mtDNA were probed for using random-primed amplified fragments of G. gallus mtDNA. 5 μl (50 μCi) of [α-32P]dCTP (3,000 Ci/mmol, Amersham Biosciences) was incubated with 50 ng of heat-denatured DNA and DNA-labeling beads (Amersham Biosciences) for 15 min at 37 °C. Probes for G. gallus mtDNA were amplified using the following primer pairs: 5′-TCAGCAACCCCTGCCTGTAATG-3′ and 5′-GGTGGAAGAACCATAACCAAATGC-3′ np 429–826 (probe 1); 5′-AGCAATCCGTTGGTCTTAGGAAC-3′ and 5′-GCGATGAGGAAGGTGAGTAGGTAG-3′ np 13,016–13,430 (probe 2); 5′-CAGGGTTGGTAAATCTTGTGCC-3′ and 5′-CGTTTGTGCTCGTAGTTCTCAGG-3′ np 1,523–1,797 (probe 3); 5′-CCGAGCGATTGAAGCCACTATC-3′ and 5′-CCTAAATGGGAGATGGATGAGAAGG-3′ np 5,390–5,779 (probe 4); and 5′-GCCTAACGCTTCAACACTCAGC-3′ and 5′-AAGGGGGGTAAACTGTCCATCCTG-3′ np 6,613–7,018 (probe 5) (see also Fig. 1C). Amplification was via the polymerase chain reaction. Start and end numbers are based on the published G. gallus mitochondrial genome sequence (18Desjardins P. Morais R. J. Mol. Biol. 1990; 212: 599-634Crossref PubMed Scopus (855) Google Scholar). Immobilized fragments of mouse mtDNA were detected by radiolabeled probes amplified via PCR, using the following pairs of primers: 5′-CAAAGGTTTGGTCCTGGCCT-3′ and 5′-TGTAGCCCATTTCTTCCCA-3′ np 69–790; 5′-CACCTTCGAATTTGCAATTCG-3′ and 5′-CTGTTCATCCTGTTCCTGCT-3′ np 5,215–5,709; 5′-CGCCTAATCAACAACCGTCT-3′ and 5′-TGGTAGCTGTTGGTGGGCTA-3′ np 8,032–8,497; and 5′-AACTGAACGCCTAAACGCAGGGA-3′ and 5′-AACTGGATTTGAAGTTGCTAGGCA-3′ np 13,867–14,518. Primer sequences were based on the original published sequence of mouse mtDNA (24Bibb M.J. Van Etten R.A. Wright C.T. Walberg M.W. Clayton D.A. Cell. 1981; 26: 167-180Abstract Full Text PDF PubMed Scopus (1359) Google Scholar). Southern hybridization was overnight at 65 °C in 0.25 m sodium phosphate, pH 7.2, 7% SDS. Post-hybridization washes were 1× SSC followed by 0.1× SSC, 0.1% SDS, both for 30 min at 65 °C. Filters were exposed to x-ray film and developed after 0.5–10 days. Free 5′ End Mapping by Ligation-mediated (LM) PCR—Approximately 0.5 μg of purified mtDNA was used as template for free 5′ end mapping. The DNA was pretreated with 8 units of λ exonuclease, according to the Replication Initiation Point mapping technique described previously (25Bielinsky A.K. Gerbi S.A. Science. 1998; 279: 95-98Crossref PubMed Scopus (131) Google Scholar), to remove spurious free 5′ ends created by adventitious DNA damage. In addition, covalently bound RNA was degraded by heating samples for 5 min at 95 °C in 0.1 m NaOH (26Abdurashidova G. Deganuto M. Klima R. Riva S. Biamonti G. Giacca M. Falaschi A. Science. 2000; 287: 2023-2026Crossref PubMed Scopus (155) Google Scholar), and after neutralization with HCl, ligation-mediated PCR was performed essentially as described by Kang et al. (27Kang D. Miyako K. Kai Y. Irie T. Takeshige K. J. Biol. Chem. 1997; 272: 15275-15279Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Briefly, initial primer extension was performed with oligonucleotide H1, 5′-TAGTCCTTGGGGTCTAACCAAGC-3′ (np 16,715–16,737). After ligation to a unidirectional linker, prepared by hybridizing oligonucleotides 5′-GCGGTGACCCGGGAGATCTGTATTC-3′ and 5′-GAATACAGATC-3′ (27Kang D. Miyako K. Kai Y. Irie T. Takeshige K. J. Biol. Chem. 1997; 272: 15275-15279Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), samples were subjected to 20 cycles of PCR, with H2, 5′-CCAAGCGGGGAATAATATGACTTAT-3′ (np 16,697–16,720), in combination with the longest linker primer. Finally, two rounds of primer extension were performed using radiolabeled H3 5′-TTAGCTGTGGCGTCTAATCCTTCG-3′ (np 16,634–16,657) or H4 5′-GGTATGGGCTAGGTTTTGTCTTGG-3′ (np 16,417–16,440). One-step sequencing reactions using 5′ end-labeled primers were used to generate sequencing ladders (28Slatko B. Albright L.M Tabor S. Ju J. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons. Inc., New York1999: 7.4A.8-7.4A.9Google Scholar). LM-PCR products and sequencing reactions were separated on 6% polyacrylamide gels containing 7 m urea. Primers corresponding to G. gallus mtDNA were based on the published sequence (18Desjardins P. Morais R. J. Mol. Biol. 1990; 212: 599-634Crossref PubMed Scopus (855) Google Scholar). Origin Detection and Mapping—In a previous study, we reported that initiation of mtDNA replication encompasses a broad zone downstream of the 3′ end of the D-loop, in mammals (10Bowmaker M. Yang M.Y. Yasukawa T. Reyes A. Jacobs H.T. Huberman J.A. Holt I.J. J. Biol. Chem. 2003; 278: 50961-50969Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Therefore, a series of fragments of chick mtDNA was separated by neutral two dimensional AGE and examined for evidence of initiation arcs, with particular emphasis on the region downstream of the 3′ end of the D-loop. Restriction sites for the fragments analyzed in this study are illustrated on a schematic map of the G. gallus mitochondrial genome (Fig. 1C). Analysis of a 7.8-kb AflIII fragment of G. gallus mtDNA, spanning np 10,540–1,603 and including OH, revealed a prominent initiation arc and a weak simple Y arc terminating in a prominent spot above the linear duplex arc (Fig. 2A, and interpreted in supplemental information). The prominent initiation arc suggests the majority of initiation events occur within the fragment, via a θ mechanism. The prominent spot above the linear duplex arc can be explained by arrest in the NCR of one of a pair of replication forks before the other fork exits the fragment at np 10,540 or by unidirectional replication in the vicinity of OH. In the former case, OH acts as the terminus of replication. The initiation arc comprises products of coupled leading and lagging-strand replication based on their mobility on two-dimensional AGE and their resistance to single strand nuclease (Fig. 2B). A shorter DraI fragment (np 14,075–988), again including OH, also gave rise to an initiation arc more prominent than the simple Y arc (Fig. 2C), whereas a 7.4-kb fragment lacking OH yielded a Y arc considerably stronger than the initiation arc (Fig. 2D). Hence, some initiation events must arise at sites other than OH, which excludes the unidirectional replication from a discrete origin (OH) as the sole mechanism of replication for G. gallus mtDNA. Still weaker initiation arcs were associated with FauI (np 8,831–16,351) and MscI (np 8,334–15,947) restriction fragments (Fig. 2, E and F) in which the OH proximal ends are more distant from OH than that of the BsmBI fragment (Fig. 2D). These data can be interpreted in one of two ways. Either G. gallus mtDNA contains a series of unidirectional origins of decreasing strength, extending from OH into the cytochrome b gene, or G. gallus mtDNA contains an initiation zone of bidirectional replication whose center lies close to the 3′ end of the D-loop region. Direction of Fork Movement—If the replication of chick mtDNA initiates bidirectionally across a zone adjacent to the 3′ end of the D-loop, and replication terminates in the NCR, then replication forks will travel in one direction only through fragments outside the initiation zone. To test this proposition, fork-direction gels were produced. These gels require an in-gel restriction enzyme digestion treatment between the first and second electrophoresis steps (23Friedman K.L. Brewer B.J. Methods Enzymol. 1995; 262: 613-627Crossref PubMed Scopus (204) Google Scholar, 29Brewer B.J. Lockshon D. Fangman W.L. Cell. 1992; 71: 267-276Abstract Full Text PDF PubMed Scopus (204) Google Scholar). Fragments from several regions of the genome, including ones distant from the 3′ end of the D-loop, gave rise to pairs of Y arcs (Fig. 3, A, D, G, and J) indicating that replication forks enter these fragments from both ends. Therefore, replication forks travel in both directions on G. gallus mtDNA. In general, one arc was more prominent than the other, and wherever this was the case it was indicative of a majority of replication forks traveling away from the 3′ end of the D-loop (Fig. 3, A, D, and G) (see supplemental information). The major arc is consistent with bidirectional replication arising from a zone proximal to the D-loop 3′ end and terminating at or near OH, so that most of the genome is replicated by the counterclockwise-moving fork. It is equally consistent with counterclockwise unidirectional replication originating in the NCR. In contrast, the other, fainter arc comprising forks traveling toward the 3′ end of the D-loop (Fig. 3, A, D, and G) fits neither model, rather it necessitates initiation at sites far from the proposed initiation zone. Unidirectional Versus Bidirectional Replication—Unidirectional replication originating in the NCR would contribute forks traveling exclusively in one direction, whereas a bidirectional initiation zone would generate forks traveling in two directions. Fork-direction analysis of a BsoBI-BsaHI fragment spanning np 12,777–15,152 indicated that most forks travel away from the 3′ end of the D-loop (Fig. 3G). In contrast, fork-direction analysis of a smaller fragment with one end closer to the 3′ end of the D-loop (MboI fragment np 14,866–16,476) revealed two simple Y arcs of approximately equal intensity (Fig. 3J). The difference between the MboI-HincII (Fig. 3J) and the BsoBI-BsaHI (Fig. 3G) fragments is decisive. It indicates that in a fragment spanning np 15,405–16,476, replication forks exit the fragment at the OH proximal end as frequently as the OH distal end, whereas in a fragment spanning np 12,777–15,152 most forks exit at the OH distal end. This is entirely consistent with bidirectional initiation at dispersed sites, whereas unidirectional replication from the NCR would yield only one simple Y arc on fork direction gels, and there would be no difference in the results from the BsoBI/BsaHI and MboI/HincII fragments. Mapping of the Initiation Zone Based upon Fork-direction Gel Data—Comparison of the set of fork-direction gels also provided an independent means of mapping the initiation zone of G. gallus mtDNA. Based on the equal intensity of the two Y arcs associated with the MboI-HincII fragment (np 15,405–16,476) (Fig. 3J), replication initiates either side of the center of the fragment (np ∼16,000) at equal frequency. That is, approximately half of all initiation events occur at least 1.8 kilobases downstream of the 3′ end of the D-loop. In contrast, in the BsoBI-BsaHI fragment spanning np 12,777–15,125, the signal from the Y arc corresponding to forks traveling away from the 3′ end of the D-loop was greater than the signal of the Y arc comprising forks traveling toward the 3′ end of the D-loop, (Fig. 3G). Therefore, most initiation events originate upstream of the center of this fragment (np ∼14,000); a conclusion fully compatible with the standard two-dimensional AGE analysis, which predicts an initiation zone adjacent to the NCR. Initiation of Replication Occurs Genome-wide in Chick mtDNA—The detection of an arc comprising forks traveling toward the 3′ end of the D-loop in fragments outside the proposed initiation zone was unexpected (Fig. 3, A, D, and G). These arcs could in theory have arisen by a quite distinct mechanism such as rolling-circle replication, which is believed to operate in yeast mtDNA (30Maleszka R. Skelly P.J. Clark-Walker G.D. EMBO J. 1991; 10: 3923-3929Crossref PubMed Scopus (139) Google Scholar, 31Ling F. Shibata T. Mol. Biol. Cell. 2004; 15: 310-322Crossref PubMed Scopus (51) Google Scholar, 32Han Z. Stachow C. Chromosoma (Berl.). 1994; 103: 162-170PubMed Google Scholar). Further analysis of fragments of chick mtDNA from around the genome revealed initiation arcs in fragments covering all regions, however (Fig. 4). Hence, θ replication can account for all the replication intermediates of chick liver mtDNA. Initiation arcs were faintest in fragments far from the initiation zone defined above, such as a DraI fragment spanning np 3,448–10,746 and an XhoI fragment spanning np 5,456–12,777 (Fig. 4, A and B).