A Bidirectional Origin of Replication Maps to the Major Noncoding Region of Human Mitochondrial DNA  Takehiro Yasukawa, Ming-Yao Yang, Howard T. Jacobs, Ian J. Holt  Molecular Cell  Volume 18, Issue 6, Pages 651-662 (June 2005) DOI: 10.1016/j.molcel.2005.05.002 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Abundant RNase H-Resistant Replication Intermediates Are Found in Human Cultured Cells Amplifying mtDNA in Contrast to Untreated Cells 2D-AGE of HincII- (A–D), DraI- (E–H), and HincII/DraI-digested (K) mtDNA from purified mitochondria of human 143B cells. (I and J) DraI-digested mtDNA from HeLa cells. (A, B, E, F, J, and K) DNA from cells amplifying mtDNA after transient drug-induced depletion (see Experimental Procedures). (C, D, G, H, and I) mtDNA from untreated cells. “+RH” indicates samples treated with RNase H. (B′, D′, F′, and H′) Illustrations of the molecular species unmodified by RNase H. Positions of relevant restriction sites are shown on a circular map of human mtDNA above the 2D gel images. The NCR and cytochrome b gene (Cyt b) are indicated by broad gray lines superimposed on the mtDNA circle, OH is the previously proposed origin of strand-asynchronous heavy-strand replication, and map positions of radiolabeled probes are indicated by broad lines inside the circle (see Experimental Procedures). Probe a (nt 16,341–151) detected the 3,939 bp HincII fragment (nt 13,636–1,006), which includes the NCR (A–D); probe b (nt 14,976–15,904) detected the 3,739 bp DraI fragment (nt 12,271–16,010) lacking the NCR (E–J) and the HincII/DraI fragment, nt 13,636–16,010 (K). The meaning of the terms upstream and downstream, used throughout the manuscript, is indicated above the map. A prominent region of the bubble arc (“i” in B′) was specifically associated with fragments containing OH (see text and Figure S6 for details). Molecular Cell 2005 18, 651-662DOI: (10.1016/j.molcel.2005.05.002) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Comparison of Bubble Arc Intensity in Different mtDNA Fragments from Placenta and Cells Amplifying mtDNA 2D-AGE of purified mtDNA from cells amplifying mtDNA: 143B (A, D, G, I, J, and L), HeLa (C and F), or from placenta (B, E, H, and K). (A) is a reproduction of Figure 1B. (D) is a longer exposure of Figure 1E. (I and L) Longer exposures of (G) and (J), respectively. Radiolabeled probe a was applied in (A)–(C), all others used probe b. Restriction fragments detected were: HincII, nt 13,636–1,006 (A)–(C); DraI, nt 12,271–16,010 (D)–(F); NciI, nt 13,365–16,454 (G)–(I); and HincII/NsiI, nt 13,636–49 (J)–(L). The very faint bubble arc detected after prolonged exposure in (I) is marked by an arrow. Molecular Cell 2005 18, 651-662DOI: (10.1016/j.molcel.2005.05.002) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Bubble Arcs Are Associated with Small NCR-Containing Fragments of Human mtDNA 2D-AGE blots of purified 143B cell mtDNA were hybridized to probe a. (A–F) DNA from cells amplifying mtDNA. (G and H) DNA from untreated cells. Restriction digests as indicated. Fragments detected were AccI, nt 15,255–1,504 (A–C, G, and H); BsaHI/HincII, nt 15,615–1,006 (D); AccI/MspA1I, nt 15,255–526 (E); and AccI/MfeI, nt 15,255–242 (F). Abbreviations: RH, treated with RNase H; SSB, incubated with single-stranded binding protein; 1n, unit length fragment; i, the region of the bubble arc close to 1n; ii, the remainder of the bubble arc; iii, paused RI on the fork arc; iv, partially single-stranded molecules. A schematic linear map shows the relevant region of human mtDNA. The broad gray line indicates the 1122 bp of the NCR (nt 16,024–576), and the black bar shows the position of the probe. The size of each fragment in bp is shown in parentheses. Molecular Cell 2005 18, 651-662DOI: (10.1016/j.molcel.2005.05.002) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 The Bubble Arcs in Small mtDNA Fragments Are Consistent with Bidirectional Replication Initiating at Sites Near One End of the NCR, Whereas OH Is the Terminus 2D-AGE blots of purified mtDNA from 143B cells amplifying mtDNA hybridized with probes c (A and B), a (C and E), or a′ (F). Fragments detected were: AccI/NsiI, nt 15,255–49 ([A], an arrow indicating the bubble arc); AccI/MfeI, nt 15,255–242, including OH (B); AccI, nt 15,255–1,504 ([C], equivalent to Figure 3A but with 40 hr recovery after drug treatment instead of the usual 24 hr, revealing a clearer double fork arc that is arrowed and schematized in [D]); AccI/DraI, nt 16,010–1,504 (E); and NciI/AccI, nt 16,454–1,504 (F). The estimated nucleotide position of the termination spots is marked on the fork arc. The map shows the relevant region of human mtDNA. Black bars indicate the positions of the probes with the size of each fragment in bp shown in parentheses. Molecular Cell 2005 18, 651-662DOI: (10.1016/j.molcel.2005.05.002) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Prominent Free 5′ Ends of DNA in the NCR of Human mtDNA LM-PCR on samples of purified human 143B cell mtDNA from untreated cells (U) or cells amplifying mtDNA (A). (A) Free 5′ ends detected by various primer sets. Clusters I and II denote two sets of prominent free 5′ ends, see text for details. Equivalent experiments for HeLa cells gave the same products (data not shown). (1H) Primer set H1/H2/H3 for the H strand in the OH region. (2H) Primer set H4/H5/H6 for the H strand in the OH-distal portion of the NCR. (3H) Primer set H4/H5/H7 for the H strand downstream of the NCR. (4L) Primer set L1/L2/L3 for the L strand in the OH-distal portion of the NCR. Other L strand primer sets for the remainder of the NCR gave only faint, heterogeneous products (data not shown). Sequence ladders run adjacent to the LM-PCR products map the position of each band at the nucleotide level (greater separation of 5A-2H is shown in Figure S10). Based on Southern blot quantification (Figure S11), the ratio of U to A mtDNA was 1:1 in all subpanels. (B) Approximate positions of LM-PCR primers (lines with half-arrowhead) aligned against a linear map of the NCR (dark gray line) and cytochrome b gene (light gray line). Closed circles indicate radiolabeled primers. (C) Signal intensity by phosphorimaging of the bands from clusters I and II. The intensity of the band at nt 191 from untreated cells was arbitrarily set at one, with that of other bands expressed relative to this value. Each column represents the mean of five experiments, and error bars are standard deviations. Molecular Cell 2005 18, 651-662DOI: (10.1016/j.molcel.2005.05.002) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Cluster II Free 5′ Ends Are More Transient Than Those of Cluster I, Particularly in Cells Amplifying mtDNA LM-PCR analysis of purified mtDNA from untreated 143B cells (U), cells amplifying mtDNA (A), cells treated with ddC (D), and linearized and gel-purified nonreplicating mtDNA (L). Clusters I and II are as described in the text. (A) H strand 5′ ends detected by primer sets H4/H5/H6 (lanes 1 and 3) or H8/H9/H6 (lanes 2 and 4). (B) The relative abundance of cluster I and cluster II 5′ ends based on phosphorimaging. Black and white bars indicate the prominent bands associated with primer set H4/H5/H6, hatched bars relate to primer set H8/H9/H6. The intensity of the band at nt 191 was arbitrarily set at one (lanes 1–4), with the intensity of other bands expressed relative to this value. (C) H strand 5′ ends detected in various DNA samples by using primer sets H1/H2/H3 (lanes 1 and 2) or H4/H5/H6 (lanes 3–6). Lanes 1 and 3, unfractionated 143B mtDNA template (U); lanes 2 and 4, linearized, nonreplicating mtDNA (L); lane 5, DNA from cells amplifying mtDNA (A); lane 6, DNA from cells treated with ddC for 72 hr with no recovery (D). 5′ ends at both clusters are diminished in nonreplicating mtDNA or mtDNA from ddC-treated cells, confirming their association with replication. Molecular Cell 2005 18, 651-662DOI: (10.1016/j.molcel.2005.05.002) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Models of mtDNA Replication in Cultured Human Cells (A–C) Bidirectional, strand-coupled replication. Synthesis initiates on the H strand most frequently at nt 16,197, as depicted in (A), or at one of the other cluster II sites (see Figure 5). Synthesis on the L strand initiates predominantly at nt 16,184. Early arrest of one replication fork (B) occurs at a termination site near OH (cluster I). H4 indicates the position of the initial primer used in the experiment of Figure 6A, lanes 1 and 3, located 0.5 kb downstream of the NCR, which detects cluster I and II 5′ ends. A primer 2.5 kb downstream of the NCR cannot bind the nascent strand at this stage. Once the nascent H strand reaches nt 13,493 (C), primer H8 can bind; however, by this stage the initiation site is no longer detectable because the first Okazaki fragment of the H strand has been synthesized at the oppositely moving fork and ligated to the 5′ end at the initiation site. In contrast, a lagging-strand initiation point opposite a leading-strand termination site creates a more persistent 5′ end (see also Figure S8C). This model can explain why primer H8, located 2.5 kb from the NCR (see Figures 6A and 6B), detects relatively more abundant free 5′ ends at cluster I than cluster II, whereas H4, which is 2 kb closer to the NCR, detects proportionately more cluster II 5′ ends. Closed black circles denote free 5′ ends of DNA; the closed black box represents the cluster of termination sites at OH. (D–F) The strand-asynchronous model. This model predicts free 5′ ends exclusively at OH on the H strand and on the L strand only at the “origin of light-strand replication,” (OL), nt 5,741–5,752. Molecular Cell 2005 18, 651-662DOI: (10.1016/j.molcel.2005.05.002) Copyright © 2005 Elsevier Inc. Terms and Conditions