Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand
2006; Springer Nature; Volume: 25; Issue: 22 Linguagem: Inglês
10.1038/sj.emboj.7601392
ISSN1460-2075
AutoresTakehiro Yasukawa, Aurelio Reyes, Tricia J. Cluett, Mingyao Yang, Mark Bowmaker, Howard T. Jacobs, Ian Holt,
Tópico(s)DNA Repair Mechanisms
ResumoArticle26 October 2006free access Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand Takehiro Yasukawa Takehiro Yasukawa MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Aurelio Reyes Aurelio Reyes MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Tricia J Cluett Tricia J Cluett MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Ming-Yao Yang Ming-Yao Yang MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Mark Bowmaker Mark Bowmaker MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Howard T Jacobs Howard T Jacobs Institute of Medical Technology and Tampere University Hospital, University of Tampere, Finland Search for more papers by this author Ian J Holt Corresponding Author Ian J Holt MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Takehiro Yasukawa Takehiro Yasukawa MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Aurelio Reyes Aurelio Reyes MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Tricia J Cluett Tricia J Cluett MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Ming-Yao Yang Ming-Yao Yang MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Mark Bowmaker Mark Bowmaker MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Howard T Jacobs Howard T Jacobs Institute of Medical Technology and Tampere University Hospital, University of Tampere, Finland Search for more papers by this author Ian J Holt Corresponding Author Ian J Holt MRC-Dunn Human Nutrition Unit, Cambridge, UK Search for more papers by this author Author Information Takehiro Yasukawa1,‡, Aurelio Reyes1,‡, Tricia J Cluett1, Ming-Yao Yang1, Mark Bowmaker1, Howard T Jacobs2 and Ian J Holt 1 1MRC-Dunn Human Nutrition Unit, Cambridge, UK 2Institute of Medical Technology and Tampere University Hospital, University of Tampere, Finland ‡These authors contributed equally to this work *Corresponding author. MRC-Dunn Human Nutrition Unit, Wellcome Trust-MRC Building, Hills Road, Cambridge CB2 2XY, UK. Tel.: +44 1223 252840; Fax: +44 1223 252845; E-mail: [email protected] The EMBO Journal (2006)25:5358-5371https://doi.org/10.1038/sj.emboj.7601392 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Using two-dimensional agarose gel electrophoresis, we show that mitochondrial DNA (mtDNA) replication of birds and mammals frequently entails ribonucleotide incorporation throughout the lagging strand (RITOLS). Based on a combination of two-dimensional agarose gel electrophoretic analysis and mapping of 5′ ends of DNA, initiation of RITOLS replication occurs in the major non-coding region of vertebrate mtDNA and is effectively unidirectional. In some cases, conversion of nascent RNA strands to DNA starts at defined loci, the most prominent of which maps, in mammalian mtDNA, in the vicinity of the site known as the light-strand origin. Introduction The most widespread mechanism of DNA replication entails concurrent leading and lagging-strand DNA synthesis (Kornberg and Baker, 1992). RNA oligonucleotides are required for multiple priming events on the lagging strand, whereas the leading strand requires only a single priming event, which may involve either RNA or protein, this asymmetry reflecting the antiparallel arrangement of double-stranded DNA (Watson and Crick, 1953). Animal mitochondrial DNAs (mtDNA) are covalently closed circular molecules of approximately 16 kb. They encode essential polypeptides of the oxidative phosphorylation system, and the ribosomal and transfer RNAs necessary for their synthesis. Replication of mammalian mtDNA was long believed to occur via continuous, but asynchronous synthesis of each strand, rather than by a conventional strand-coupled mechanism. The model was initially elaborated on the basis of electron microscopy, which revealed partially single-stranded replication intermediates (RIs) (Robberson et al, 1972). Prominent 5′ ends were mapped to two sites: those on the putative leading (heavy) strands to a specific area of the major non-coding region (NCR) (Crews et al, 1979), designated the origin of heavy strand replication (OH), and those on the presumed lagging-strand to a short spacer region in a cluster of five tRNA genes (Kang et al, 1997; Tapper and Clayton, 1982), named the origin of light-strand replication (OL). Many molecules of mammalian mtDNA contain a short triple-stranded region, or D-loop (Arnberg et al, 1971; Kasamatsu et al, 1971). The third strand of the D-loop, 7S DNA, is readily displaced by heating, it spans much of the NCR and its 5′ end coincides with OH. This led to the proposal that 7S DNA represented stalled or aborted RIs. More recently, RIs with properties of conventional strand-coupled DNA synthesis, but inconsistent with the strand-asynchronous model, were detected in mammalian mitochondria using two dimensional agarose gel electrophoresis (2D-AGE) (Holt et al, 2000). These were shown to initiate across a broad zone (ori-Z) spanning several kilobases in solid tissues (Bowmaker et al, 2003), but in a narrower zone within the NCR in cultured cells re-amplifying mtDNA after drug-induced mtDNA depletion (Yasukawa et al, 2005). In addition, RIs with extensive RNA patches on the L-strand were detected (Yang et al, 2002). The latter were demonstrated to be labile to RNA loss during extraction, potentially accounting for the partially single-stranded mitochondrial RIs detected previously by electron microscopy (Robberson et al, 1972), and again recently by atomic force microscopy (AFM) (Brown et al, 2005). In the present study, we set out to determine the precise structure of ribonucleotide-rich RIs. Remarkably, we found that RNA can extend across one entire nascent strand of mtDNA, prior to lagging strand DNA synthesis. Results Replicating circles of mtDNA after restriction digestion are indicative of ribonucleotide incorporation at sites across much of the mitochondrial genome The existence of RNA patches on the L-strand of replicating mtDNA (Yang et al, 2002) predicts one branch of a replication bubble will not be cleaved by a restriction enzyme, wherever the site coincides with an RNA patch. For restriction enzymes cutting mtDNA at one site only, ribonucleotide blockage will produce replicating circles of mtDNA, analogous to uncut circular molecules with a broken bubble (Lucas et al, 2001; Martin-Parras et al, 1992, 1998). After digestion of chick liver mtDNA with SfoI, which cuts exclusively at nucleotides (nt) 15 153, two major arcs of RIs were apparent a double Y arc and an arc of replicating circles, or ‘eyebrow’ (Figure 1(1), and interpreted in Figure 1(5)). Cleavage on the other side of the major NCR at nt 1879, gave a quite different pattern of RIs, there was a prominent, extensive bubble arc consistent with a theta mechanism of replication, and very few uncut circles (Figure 1(2), and interpreted in Figure 1(6)). An eyebrow was again evident when the DNA was digested with restriction enzymes cutting uniquely at nt 12 549 or 10 885 (Figure 1(3 and 4), respectively) indicating that these sites are also frequently blocked in replicating molecules. Analysis of mouse liver mtDNA produced similar results; BglII cutting once close to the cytochrome b gene end of the mouse NCR yielded a prominent eyebrow (Figure 1(7)) as did another enzyme cutting 2 kb away in the coding region (Figure 1(8)); the eyebrow was significantly shorter yet still prominent when mouse liver mtDNA was digested with NcoI, which cuts over 6 kb downstream of the NCR (Figure 1(9)). Digesting mouse mtDNA with PstI, which cuts duplex DNA at two sites also yielded an eyebrow (Figure 1(10)), which is the result of restriction site blockage at both nts 12 243 and 8424 on the same branch of a replicating molecule. Cleavage of mouse mtDNA at nts 14 829, 9215 and 5369 yielded a much weaker eyebrow (Figure 1(11)), whereas no appreciable eyebrow was associated with MluI digested mouse mtDNA cut at nt 1771 (Figure 1(12)). Eyebrows were evident in chick mtDNA after digestion with enzymes expected to cleave at nts 3512, 2843 and 1600 among other sites (Supplementary Figure 1(1–3)). RNase H treatment modified eyebrows (Supplementary Figure 1(5–8)), consistent with the idea that the failure to cleave at the various restriction sites is attributable to ribonucleotide incorporation. Figure 1.Restriction site blockage in mtDNA of chick and mouse. Schematic maps of Gallus gallus and Mus musculus mitochondrial genomes, with restriction sites and probes, appear at the top of the figure. The position of the site designated as the heavy-strand origin of replication (OH) and the major NCR are marked on the maps together with the positions of the cytochrome b (Cyt b) and 12S rRNA genes (12S rRNA). ‘Downstream’ refers to events and loci on the Cyt b gene side of OH, whereas ‘upstream’ refers to the 12S rDNA side of OH. 2D-AGE gels are as follows: Panels 1–4, chick liver mtDNA cleaved with the restriction enzymes indicated and hybridized with probe c1. Panel 5, interpretation of panel 1, SfoI-cuts chick liver mtDNA at a single site in the genome, at nt 15 153. The short length and low trajectory of the bubble arc (b) rule out a bidirectional origin near the center of the genome-length fragment ∼nt 9000, since this would produce a rather similar pattern to the ClaI digest (Panel 2, and interpreted in panel 6). Instead, to reconcile both patterns of intermediates (panels 1 and 2), the origin must lie close to one end of the fragment, and must also be coincident or adjacent to the definitive terminus, as illustrated (panels 5 and 6). Cleavage of a bubble on both branches will convert a replicating circle to a linear molecule with a fork at either end. In the case of SfoI-digested chick liver mtDNA, the bubble arc is short; hence, the restriction site must lie close to the origin; moreover, most of the molecule must be copied by a single active fork, as two active forks would produce a double Y arc ending near the apex of an X arc (well above the linear duplex arc). Thus, the prominent Y-like arc (panel 1) must be a double Y arc (dY), with a static fork close to one end of the fragment (see also (Schvartzman et al, 1993). The other prominent arc (e) lying above the position of a standard Y arc is inferred to be an arc of circular molecules of increasing size (an eyebrow), as it corresponds approximately to previously described arcs of replicating circular molecules (Brewer et al, 1988; Belanger et al, 1996). As illustrated, it is a predicted product of site blockage (red cross) on one branch of a theta structure. The position of open/nicked circular mtDNA (c) was determined by separating uncut mtDNA by 2D-AGE (data not shown). Panel 6: interpretation of panel 2. The unique ClaI site lies on the other side of the NCR from the SfoI site at nt 1879, in chick mtDNA. A small amount of uncut circles (c) remain. The bubble arc (b) extends over almost the entire length of the fragment, with a short double Y arc (dY) and a faint Y arc also visible. This is consistent with unidirectional replication originating from a site near the (Cyt b gene) end of the fragment, as illustrated. A bidirectional terminus near one end of the fragment, adjacent to an initiation zone, would give the same pattern. No eyebrow arc was evident; however, in the case of ClaI such an arc would be short, corresponding to only 6% of a full-length eyebrow arc and could well coincide with the bubble arc; moreover, as it represents the final stages of replication it might be short-lived. Panels 7–12, mouse liver mtDNA cut with the restriction enzymes indicated, and hybridized with probe m7. Site coordinates are either the unique sites of digestion, or (panels 10 and 11) the site of digestion furthest from the Cyt b end of the D-loop, as this is indicative of the distance travelled (clockwise) by a unidirectional fork starting from the NCR, see also panel 13. 1n—unit length fragment, b—bubble arc, dY—double Y arc, c—circular monomers, e—eyebrow arc, cd—circular dimers (the end of an eyebrow arc). Eyebrows correspond to replicating circular molecules, and have previously been associated with both theta and rolling circle replication (Belanger et al, 1996; Preiser et al, 1996; Brewer et al, 1988). Panel 13—Interpretation of the 2D-AGE data, consistent with a unidirectional origin or terminus at OH and restriction site blockage (indicated by a red cross) at several points on one strand of replicating mtDNA molecules. Cyan bars indicate restriction sites that the advancing replication fork has not yet reached, generating bubble arcs. (For colour figure see online.) Download figure Download PowerPoint Mapping the zone of ribonucleotide incorporation using truncated eyebrows Unlike conventional eyebrows, those associated with mtDNA replication migrated on 2D gels with a gap of variable size visible between simple circles and the start of the eyebrow (Figure 1(1–4) and (7–9)). The size of the gap is predicted to correspond with the distance the replication fork(s) has travelled before reaching the restriction site and thereby provides information on the origin, direction and terminus of replication (schematically illustrated in Figure 1(13)). In mouse, the gap between eyebrow and circular monomers was shortest in the case of BglII (Figure 1(7)), whose one site in the mouse mitochondrial genome lies just beyond the NCR. XhoI cuts approximately 2 kb from the NCR and here the gap was noticeably larger (Figure 1(8)); digestion with NcoI, cleaving 6 kb downstream of the NCR, eliminated approximately half the eyebrow (Figure 1(9)). These results map the start of the zone of ribonucleotide incorporation close to the BglII site, extending around a substantial portion of the genome. Note also that the distribution of digestion sites generating prominent bubble arcs and double-Y arcs is also consistent with initiation and termination of replication in the region where ribonucleotide incorporation begins, although these data alone cannot discriminate between unidirectional replication initiating in the NCR, upstream of nt 15 336, and bidirectional replication initiating close to the NCR accompanied by fork arrest and termination at or close to OH. Slow-moving Y-like arcs reveal the fine distribution of ribonucleotide-blocked restriction sites in the genome We next considered the expected outcome of restriction site blockage on RIs cut additionally within the region not yet replicated. If an advancing replisome incorporates ribonucleotides at a single site on one nascent strand and the flanking sites are cleaved, this will result in two fragments remaining joined, thereby creating a slow moving Y-like arc (SMY, species B, Figure 2(1) and arc B in Figure 2(2)). If, however, the adjacent fragment contains a unidirectional origin (or terminus), the RI will contain a second, static, branch (species C and D, Figure 2(1)), altering the size and mobility of the RIs and resulting in a distinct type of SMY arc that never meets the linear diagonal (fSMY, flying SMY arc). The length of the static branch, in conjunction with its position within the fragment, determines how close to the linear arc the fSMY arc begins and ends. A terminus or unidirectional origin close to one end of the restriction fragment (Figure 2(1-C)) is predicted to form an arc close to the linear duplex arc (Figure 2(2, arc C)), whereas an origin located centrally within a fragment (Figure 2 (1-D)), will yield an arc well above the linear duplex arc (Figure 2(2), arc D). Analysis of digests carried out with restriction enzymes cutting chick mtDNA twice or more demonstrated that all fragments immediately adjacent to the cytochrome b end of the NCR produced fSMY arcs (slow-moving arcs which both begin and end at positions above the linear duplex arc), indicating blocked sites at nts 11 720, 12 777, 14 017, 15 403, 15 405, 15 947 (Figure 2(3–8)) and 16 645 (Supplementary Figure 2(1)). Note that the fSMY arc moves inexorably closer to the linear arc as the restriction site approaches the NCR (nts 1–1227); that is, as the static branch shortens (Figure 2(3–8), and Supplementary Figure 2(1)). These results are in agreement with the eyebrow data, mapping the unidirectional origin (or terminus) of ribonucleotide-rich replication close to the NCR ( 400 nts
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