Mechanism of mitochondrial DNA replication in mouse L-cells: Kinetics of synthesis and turnover of the initiation sequence

Bogenhagen, Daniel F.; Clayton, David A.

doi:10.1016/0022-2836(78)90269-3

Cited by 111 publications

(49 citation statements)

References 14 publications

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“…L-cells complete mtDNA strand synthesis in about an hour. 18 In contrast, it has been estimated that complete replication of mtDNA requires about 3 hours in rat heart, 12 hours in kidney, 15 hours in brain, and 30 hours in liver. 17 Although replication rates in these tissues were not directly measured, these estimates may provide insight into the mechanism for the alternative O L usage.…”

Section: Light-strand Origins Of Replicationmentioning

confidence: 99%

Genesis and Wanderings: Origins and Migrations in Asymmetrically Replicating Mitochondrial DNA

Brown¹,

Clayton²

2006

Cell Cycle

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Mammalian mitochondria maintain a small circular genome that encodes RNA and polypeptides that are essential for the generation of ATP through oxidative phosphorylation. The mechanism of replication of mammalian mitochondrial DNA (mtDNA) has recently been a topic of controversy. New evidence has led to a modified strand-displacement model that reconciles much of the current data. This revision stems from a new appreciation for alternative light-strand origins. We consider here some of the potential mechanisms for light-strand origin initiation. We also consider further the susceptibility of branch migration within replicating mtDNA molecules. The existence of alternative light-strand origins and a propensity for branch migration in replicating mtDNA molecules exposes a new array of possible configurations of mtDNA. The assortment and assignment of these forms is relevant to the interpretation of experimental data and may also yield insight into the molecular basis of replication errors.

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Section: Light-strand Origins Of Replicationmentioning

confidence: 99%

Genesis and Wanderings: Origins and Migrations in Asymmetrically Replicating Mitochondrial DNA

Brown¹,

Clayton²

2006

Cell Cycle

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“…For example, over 95% of the newly synthesized H strand is a D-loop in the case of mouse L cells (4). This makes it difficult to precisely determine origins of the true replication form (nascent H strand) (5).…”

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In Vivo Determination of Replication Origins of Human Mitochondrial DNA by Ligation-mediated Polymerase Chain Reaction

Kang

Miyako

Kai

et al. 1997

Journal of Biological Chemistry

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A large part of replication is aborted in human mitochondria, the result being a D-loop. As few attempts have been made to distinguish free 5 ends of true replicate from those of abortive ones, we examined the 5 ends of true replicate of human mitochondrial DNA at one nucleotide resolution in vivo by making use of ligation-mediated polymerase chain reaction. The distribution and relative amounts of origins of the true replicate are exactly the same as those of total newly synthesized heavy strands, which means that the abortion of replication is independent of 5 ends. Treatment of DNA with RNase H frees 5 ends on both heavy and light strands. This is the first in vivo evidence for covalently attached primer RNA to nascent strand in human mitochondrial DNA.Replication of mitochondrial DNA (mtDNA) begins with synthesis of the heavy strand (H strand) 1 from the replication origin O H , following transcription of the light strand (L strand). Transcription of the L strand is regulated by the L strand promoter (LSP) sequence. When synthesis of the H strand has proceeded about two-thirds, synthesis of the L strand begins from replication origin O L . Thus, the total replication rate of mtDNA is determined by H strand synthesis (for reviews, see Refs. 1-3). The multiple replication origins of human mitochondrial H and L strands have been determined as free 5Ј ends of mtDNA extracted from prepared mitochondria. The molecular mechanism and physiological significance of these events are poorly understood.A large part of the synthesis of H strand is aborted, leaving a displacement loop ((D-loop) or 7 S DNA). For example, over 95% of the newly synthesized H strand is a D-loop in the case of mouse L cells (4). This makes it difficult to precisely determine origins of the true replication form (nascent H strand) (5). Few investigators have distinguished the free 5Ј end of the true replicate form from that of the D-loop even though selective detection of the free 5Ј end of the true replicate form is critical to determine replication origin. Although the 5Ј ends of the nascent H strand are suggested to be the same as those of D-loop in human mitochondria (5), it is uncertain whether the distribution and relative amount of the 5Ј ends of nascent H strand are exactly the same as those of the D-loop. In addition, the free 5Ј ends of human mtDNA are not set at one nucleotide resolution (6). To precisely determine the replication origin but not the simple free 5Ј end, it is required to selectively detect the replication origin at a higher resolution.We made use of the ligation-mediated polymerase chain reaction (LMPCR) to detect free 5Ј ends (7,8), an approach which makes feasible use of total DNA extracted directly from whole cells. By selective amplification of nascent H (not D-loop), we determined precise and comprehensive replication origins of the H strand in vivo. Here, we describe sites of free 5Ј ends and the transition sites of RNA to DNA for both H and L strands at one nucleotide resolution.

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“…H-strand DNA elongation continues around the entire length of the genome, with L-strand DNA synthesis beginning at a separate unidirectional origin (OL) only after displacement of the template strand. Interestingly, only a few initiation events at OH actually result in the synthesis of a full-length genome; nearly 95% are terminated at specific sites, depending on the vertebrate species, 500 to 1,000 bases downstream (5). This finding suggests that the site where H-strand DNA synthesis is either prematurely terminated or * Corresponding author.…”

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Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region.

Madsen¹,

Ghivizzani²,

Hauswirth³

1993

Mol. Cell. Biol.

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A methylation protection assay was used in a novel manner to demonstrate a specific bovine proteinmitochondrial DNA (mtDNA) interaction within the organelle (in organello). The protected domain, located near the D-loop 3' end, encompasses a conserved termination-associated sequence (TAS) element which is thought to be involved in the regulation of mtDNA synthesis. In vitro footprinting studies using a bovine mitochondrial extract and a series of deleted mtDNA templates identified a -48-kDa protein which binds specifically to a single TAS element also protected within the mitochondrion. Because other TAS-like elements located in close proximity to the protected region did not footprint, protein binding appears to be highly sequence specific. The in organello and in vitro data, together, provide evidence that D-loop formation is likely to be mediated, at least in part, through a trans-acting factor binding to a conserved sequence element located 58 bp upstream of the D-loop 3' end. (14). Consistent with this suggestion, closely related terminationassociated sequences (TASs) have been identified at similar positions relative to mapped D-loop DNA 3' ends in Xenopus laevis (15), cow, pig (34), and several primates (21).The current picture, therefore, is that conserved sequence domains (TAS elements) located short distances upstream of 3' ends of D-loop DNA exist in all vertebrate species and are candidate cis elements for the regulation of H-strand DNA synthesis. However, no experimental evidence regarding the potential function of TAS elements exists. Additionally, it remains unclear how such elements might function as terminators of H-strand DNA synthesis both because the number of consensus TAS domains can exceed the number of 3' D-loop termini and because TASs are found at variable distances (20 to 80 bp) upstream of these termination sites.In this study, we have adapted the methylation protection technique to assay purified bovine mitochondria for specific protein-mtDNA interactions near the D-loop 3' end in an in vivo-like environment. By this method, we identified a single protected sequence element located 58 bases upstream from the D-loop 3' end. Using partially purified protein extracts from bovine mitochondria, a 48-kDa protein(s) which binds specifically to this element was identified. In vitro DNase I footprint experiments using a series of deleted target sequences demonstrated that although seven TAS-like sequences exist in the region, only one is selectively bound. These results suggest that D-loop termination and, subsequently, mtDNA copy number may be regulated through the binding of a sequence-specific protein factor.

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Mechanism of mitochondrial DNA replication in mouse L-cells: Kinetics of synthesis and turnover of the initiation sequence

Cited by 111 publications

References 14 publications

Genesis and Wanderings: Origins and Migrations in Asymmetrically Replicating Mitochondrial DNA

Genesis and Wanderings: Origins and Migrations in Asymmetrically Replicating Mitochondrial DNA

In Vivo Determination of Replication Origins of Human Mitochondrial DNA by Ligation-mediated Polymerase Chain Reaction

Protein binding to a single termination-associated sequence in the mitochondrial DNA D-loop region.

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