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Review
. 2018 Sep 1;164(3):183-193.
doi: 10.1093/jb/mvy058.

An overview of mammalian mitochondrial DNA replication mechanisms

Takehiro Yasukawa  1 , Dongchon Kang  1
Affiliations
Review

An overview of mammalian mitochondrial DNA replication mechanisms

Takehiro Yasukawa et al. J Biochem. .

Abstract

While the majority of DNA is enclosed within the nucleus, the mitochondria also contain their own, separate DNA, the mitochondrial DNA (mtDNA). Mutations in mtDNA are associated with various human diseases, demonstrating the importance of mtDNA. Intensive studies over the last 18 years have demonstrated the presence of two distinct classes of mtDNA replication intermediates in mammals. One involves leading-strand DNA synthesis in the absence of synchronous lagging-strand DNA synthesis. Currently there are competing models in which the lagging-strand template is either systematically hybridized to processed mitochondrial transcripts, or coated with protein, until the lagging-strand DNA synthesis takes place. The other class of mtDNA replication intermediates has many properties of conventional, coupled leading- and lagging-strand DNA synthesis. Additionally, the highly unusual arrangement of DNA in human heart mitochondria suggests a third mechanism of replication. These findings indicate that the mtDNA replication systems of humans and other mammals are far more complex than previously thought, and thereby will require further research to understand the full picture of mtDNA replication.

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Figures

Fig. 1
Fig. 1
Staining image and genetic map of human mtDNA. (A) Fluorescent microscopic images of mtDNA (small green dots) (top) and mitochondria (red tubes) (middle) of culture human cells. The merged image is shown (bottom). (B) Schematic presentation of human mtDNA. Gene coding regions for proteins, ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) are shown as blue, orange and red thick lines, respectively. Encoded proteins are ND1, 2, 3, 4, 4L, 5 and 6 subunits of NADH-ubiquinone oxidoreductase (Complex I); cytochrome b (cyt b), a subunit of ubiquinone-cytochrome c oxidoreductase (Complex III); COXI, II and III, subunits of cytochrome c oxidase (Complex IV); A6 and 8, subunits of ATP synthase (Complex V). The 12S rRNA and 16S rRNA are mitochondrial rRNA genes. Mitochondrial tRNA genes are shown by their cognate amino acids with single letter notation. The position of the major noncoding region (NCR) is shown as black lines between tRNAPhe and tRNAPro genes. Transcription from the light (L)-strand promoter (LSP) is responsible for ND6 mRNA and 8 tRNAs. While transcription from the heavy (H)-strand promoter 1 (HSP1) generates 2 rRNAs and 2 tRNAs and terminates at the 3′ end of the 16S rRNA gene, transcription from HSP2 produces almost the entirety of the H-strand transcripts. OH and OL are the major replication initiation sites of H- and L-strands, respectively, under strand-asynchronous mtDNA replication models. The approximate position of OH on mtDNA is shown as an orange oval, representing the unidirectionality of this origin. Ori-b is an initiation region that appears to function as the second initiation site for unidirectional asynchronous synthesis of the H-strand and as a bidirectional initiation origin. The approximate position of Ori-b is shown as an orange/blue oval representing the uni- and bidirectionality of this origin. Conserved sequence block II (CSB II) is shown as a pale-grey rectangle. The position of 7S DNA is shown as a grey bar outside mtDNA. Approximate distances between LSP and CSB II, CSB II and OH and OH and Ori-b in human mtDNA are shown with grey italic numbers.
Fig. 2
Fig. 2
Principles of neutral/neutral two-dimensional agarose gel electrophoresis (2D-AGE). (A) Schematic diagram of 2D-AGE. (i) DNA is digested into appropriately sized fragments using restriction enzyme(s) and is run on a first-dimension gel. (ii) Then, the gel lane is excised and a second-dimension gel containing ethidium bromide (EtBr) is cast around the gel slice. (iii) After electrophoresis, the gel is blotted onto a solid support and DNA fragments of interest are visualized using Southern hybridization. (B–D) Schematic drawings of RIs. (B) Replication initiates at an origin (grey arrowhead) located in the middle of a DNA fragment (0) and progresses bidirectionally (1→3), forming a series of bubble structure molecules until the bubble ‘bursts’ as the replication forks exit the fragment. Restriction digest sites are indicated with vertical blue lines. A complete bubble arc (or initiation arc) is generated from the series of such molecules in 2D-AGE [1→3 in A-(iii)]. (C) Replication is initiated outside the fragment and the replication fork traverses from one end to the other, forming a series of ‘Y’ structure molecules (4→6) which generate a Y arc (or fork arc) in 2D-AGE [4→6 in A-(iii)]. (D) When replication is initiated and proceeds unidirectionally from an origin near the end of the fragment (3′), a bubble structure will be formed before the replisome reaches a restriction site. Then, the presence of ribonucleotides [the ribonucleotide-incorporated strand is represented as orange lines] at a restriction site blocks restriction enzyme digestion (indicated as an asterisk), giving rise to a series of molecules with one moving replication fork and one static fork. After the replisome goes beyond the restriction site, slow-moving Y-like (SMY) arcs will be formed (7→9). SMY arcs are generated [7→9 in A-(iii)] from the blockage of more than one restriction sites [see details in (45)].
Fig. 3
Fig. 3
mtDNA RIs are composed of two classes of molecules with different sensitivity to nucleases. (A) 2D-AGE analysis of OH-containing fragments of mouse liver mtDNA digested with the restriction enzyme BclI and treated with nucleases as follows: left untreated (U) or treated with low levels of RNase H (↓RH), standard levels of RNase H (RH) or RH and S1 nuclease (S1) [Figure 5 in (45) for details]. These panels are reuse of those presented in Yasukawa et al. (45). Interpretations of arcs visualized using Southern hybridization are shown below each panel. Prominent bubble arcs (bubble) and SMY arcs (SMYs) were modified by RNase H and were degraded with the addition of S1 nuclease, suggesting that they are ribonucleotide-containing RIs. On the other hand, a fraction of the bubble arcs and Y arcs were resistant to nuclease treatments, indicating that such RIs have properties of those from conventional coupled leading- and lagging-strand DNA synthesis. Nuclease-resistant bubble arcs were also detected from non-NCR-containing fragments. 1N indicates non-replicating fragments. (B) Schematic drawings of proposed molecular structures of arcs observed in (A). ‘Orange arcs’ and ‘green and black arcs’ indicate arcs drawn in orange colour and arcs in green and black colours in (A), respectively.
Fig. 4
Fig. 4
Proposed models of mtDNA replication. (A, C) Bootlace strand-asynchronous replication (Bootlace-SA replication). Replication of this mode initiates with the synthesis of an H-strand at one of two sites, OH or Ori-b. H-strand DNA synthesis (leading-strand synthesis) proceeds unidirectionally with concurrent incorporation of RNA into the lagging strand. The RNA lagging strand entails hybridization (‘threading’) of processed mitochondrial transcripts to the parental H-strand, as illustrated in (C). Delayed L-strand DNA synthesis is initiated frequently, but not exclusively at OL in mammals and proceeds unidirectionally. RNA lagging strands are removed in this process. (B, D) Strand-coupled DNA replication (SCD replication). Most of the replication within this mechanism initiates from a broad zone of several kilobases, including the gene-encoding region of mtDNA (Ori-z). In this replication mode, replication is bidirectional and the OH region appears to function as a replication fork barrier. Characterization of RIs from this process suggests that syntheses of the leading and lagging strands are synchronous (coupled) and both strands are essentially composed of DNA. The mechanism of lagging-strand synthesis remains to be elucidated. A proposed model is that lagging strands are formed with multiple short DNA fragments (Okazaki fragments) which are primed by an as yet unidentified mitochondrial primase as illustrated in (D), similar to nuclear DNA replication.
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