Reactome | Strand-asynchronous mitochondrial DNA replication

Strand-asynchronous mitochondrial DNA replication

Stable Identifier

R-HSA-9913635

DOI

10.3180/R-HSA-9913635.1

Type

Pathway

Species

Homo sapiens

Synonyms

Strand-displacement mitochondrial DNA replication

ReviewStatus

5/5

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Strand-asynchronous mitochondrial DNA replication

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The human mitochondrial genome is a circular double-stranded DNA of 16569 bp that encodes 2 rRNAs, 22 tRNAs, and 13 proteins. Based on density in a denaturing cesium chloride gradient, a heavy (H) strand and a light (L) strand are distinguishable. Two basic mechanisms of mitochondrial DNA replication have been proposed: (1) strand-asynchronous DNA replication (also called strand-displacement DNA replication), in which initiation of H strand synthesis significantly precedes initiation of L strand synthesis and each initiation is primed at a specific origin, and (2) strand-synchronous DNA replication, also called strand-coupled DNA replication, in which initiations of H strand synthesis and L strand synthesis occur concurrently and are primed by short RNA primers distributed through the genome (Yang et al. 2002, reviewed in Falkenberg 2018, Yasukawa and Kang 2018, Shokolenko and Alexeyev 2022, Falkenberg et al. 2024). Both types of synthesis may occur in mitochondria and the choice of synthesis type may depend on the abundance of the DNA helicase TWINKLE (TWNK, PEO1) and RNA transcripts (Cluett et al. 2018). The strand-asynchronous model has existed longer and has been more completely characterized (Uhler and Falkenberg 2021, Kosar et al. 2021). There is also some question of the evidence supporting the existence of RNA primers throughout the genome (Brown et al. 2005).
In the strand-asynchronous mechanism, the H strand is polymerized first from an origin of replication (OriH) located near the L strand promoter (LSP), which initiates transcription of the L strand as a template and a RNA corresponding to the sequence of the H strand as a product (Kang et al. 1997, Agaronyan et al. 2015, reviewed in Kasiviswanathan et al. 2012). The transcription elongation factor TEFM acts as a switch between transcription of the entire L strand and synthesis of a short primer for DNA replication (Agaronyan et al. 2015). The mitochondrial RNA polymerase POLRMT at LSP initiates synthesis of the H strand by polymerizing a short RNA of about 120 nucleotides that extends from the LSP to conserved sequence block 2 (CSB2) (Chang and Clayton 1985, Pham et al. 2006). The 3' end of the RNA is located in a G-quadruplex secondary structure that renders the 3' hydroxyl inaccessible for priming DNA synthesis and that creates a persistent R loop (Xu and Clayton 1996, Wanrooij et al. 2010, Wanrooij et al. 2012). RNASEH1 cleaves the RNA in the RNA-DNA duplex and creates accessible 3' hydroxyl groups (Posse et al. 2019, Misic et al. 2022).
The DNA helicase TWINKLE (TWNK, PEO1) and the mitochondrial DNA polymerase POLgamma, a complex comprising one subunit of POLG and two subunits of POLG2 (POLG:POLG2), bind the OriH region (Jemt et al. 2011, Jemt et al. 2015, Korhonen et al. 2004). TWNK binds as an open hexameric ring that closes around the DNA and hydrolyzes ATP to dissociate double-stranded DNA (Jemt et al. 2011, Jemt et al. 2015, Kaur et al. 2020, Kaur et al. 2021, reviewed in Peter and Falkenberg 2020) ahead of POLgamma, which uses the 3' hydroxyl groups of the RNA at OriH to begin polymerizing the nascent H strand (Johnson et al. 2000, Korhonen et al. 2004, Wanrooij et al. 2008, Plaza-G A et al. 2023). As polymerization proceeds, the parental H strand is displaced and bound by single strand binding protein 1 (SSBP1) (Miralles Fusté et al. 2014, Kaur et al. 2018, Plaza-G A et al. 2023). There is also evidence of long RNAs binding the displaced H strand (the "bootlace" model, Reyes et al. 2013).
Synthesis of the H strand continues until POLgamma passes the origin of L strand replication (OriL) about two thirds of the way around the 16569 bp genome. OriL becomes single-stranded and assumes a secondary loop structure that binds POLRMT, which synthesizes a short RNA that acts as a primer for DNA synthesis (Wanrooij et al. 2008, Fusté et al. 2010, Sarfallah et al. 2021). Significantly, OriL is required for mitochondrial maintenance in mice, evidence that the strand-asynchronous replication mechanism is essential (Wanrooij et al. 2012).
As POLgamma nears completion of the H and L strands, it migrates around the circular genome and reaches the 5' ends of the RNA primers. RNASEH1 cleaves all but two ribonucleotides from the primers in the nascent H and L strands (Ruhanen et al. 2011, Al-Behadili et al. 2018, reviewed in Uhler and Falkenberg 2015). Subsequent processing of the H and L strands differs slightly. A flap structure appears to be created by displacement at the 5' end of the H strand and the flap is removed by MGME1 (Uhler et al. 2016). A similar flap structure at the 5' end of the L strand is removed by another nuclease (Al-Behadili et al. 2018) that, based on in vitro evidence, may be EXOG (Wu et al. 2019, Karlowicz et al. 2022).
The remaining gaps in the H and L strands are ligated by the mitochondrial isoform of ligase III (LIG3-1) (Ruhanen et al. 2011). The resulting two double stranded mitochondrial genomes remain catenated by single strands which are resolved by topoisomerase 3A (TOP3A) (Nicholls et al. 2018).

Literature References

PubMed ID	Title	Journal	Year
18685103	Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro Falkenberg, M, Shi, Y, Farge, G, Fusté, JM, Wanrooij, S, Gustafsson, CM	Proc Natl Acad Sci U S A	2008
30102370	A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL Falkenberg, M, Reyes, A, Berglund, AK, Peter, B, Wanrooij, S, Uhler, JP, Doimo, M, Al-Behadili, A, Zeviani, M	Nucleic Acids Res	2018
34604445	Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase Kaur, P, Copeland, WC, Wang, H, Longley, MJ, Pan, H	Bio Protoc	2021
36744436	Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB Cao-García, FJ, Ibarra, B, Crespo, R, Truong, TQ, Ciesielski, GL, Lemishko, KM, Plaza-G A, I, Kaguni, LS	Nucleic Acids Res	2023
16790426	Conserved sequence box II directs transcription termination and primer formation in mitochondria Pham, XH, Falkenberg, M, Gaspari, M, Shi, Y, Farge, G, Gustafsson, CM	J Biol Chem	2006
35819194	In vitro reconstitution reveals a key role of human mitochondrial EXOG in RNA primer processing Szymanski, MR, Dubiel, AB, Krol, E, Bledea, A, Szczesny, RJ, Szczesny, B, Purzycki, P, McAuley, RJ, Czerwinska, J, Brzuska, G, Grzelewska, M, Karlowicz, A	Nucleic Acids Res	2022
30949702	A unique exonuclease ExoG cleaves between RNA and DNA in mitochondrial DNA replication Wu, CC, Lin, JLJ, Yuan, HS, Yang-Yen, HF	Nucleic Acids Res	2019
20129056	Mitochondrial RNA polymerase is needed for activation of the origin of light-strand DNA replication Falkenberg, M, Cluett, TJ, Jemt, E, Holt, IJ, Granycome, CE, Shi, Y, Fusté, JM, Wanrooij, S, Gustafsson, CM, Atanassova, N	Mol Cell	2010
21840902	The mitochondrial DNA helicase TWINKLE can assemble on a closed circular template and support initiation of DNA synthesis Bäckström, S, Falkenberg, M, Holmlund, T, Jemt, E, Farge, G, Gustafsson, CM	Nucleic Acids Res	2011
30605451	RNase H1 directs origin-specific initiation of DNA replication in human mitochondria Falkenberg, M, Reyes, A, Clausen, AR, Posse, V, Uhler, JP, Gustafsson, CM, Al-Behadili, A, Zeviani, M	PLoS Genet	2019
15167897	Reconstitution of a minimal mtDNA replisome in vitro Pham, XH, Falkenberg, M, Pellegrini, M, Korhonen, JA	EMBO J	2004
32213598	Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase Kaur, P, Copeland, WC, Wang, H, Longley, MJ, Wang, W, Pan, H, Countryman, P	J Biol Chem	2020
35947649	Mammalian RNase H1 directs RNA primer formation for mtDNA replication initiation and is also necessary for mtDNA replication completion Valentino, ML, Filipovska, A, Caporali, L, Larsson, NG, Filograna, R, Jiang, M, La Morgia, C, Koolmeister, C, Misic, J, Falkenberg, M, Clausen, AR, Milenkovic, D, Carelli, V, Nicholls, TJ, Jenninger, L, Jiang, S, Siira, SJ, Wredenberg, A, Xie, X, Al-Behadili, A	Nucleic Acids Res	2022
26253742	Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element Falkenberg, M, Dávila López, M, Freyer, C, Jemt, E, Shi, Y, Samuelsson, T, Uhler, JP, Gustafsson, CM, Mehmedovic, M, Persson, Ö	Nucleic Acids Res	2015
21878356	Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells Yasukawa, T, Ushakov, K, Ruhanen, H	Biochim Biophys Acta	2011
25635099	Mitochondrial biology. Replication-transcription switch in human mitochondria Temiakov, D, Morozov, YI, Agaronyan, K, Anikin, M	Science	2015
38594940	Replication and Transcription of Human Mitochondrial DNA Falkenberg, M, Larsson, NG, Gustafsson, CM	Annu Rev Biochem	2024
30256971	Single-molecule DREEM imaging reveals DNA wrapping around human mitochondrial single-stranded DNA binding protein Kaur, P, Copeland, WC, Wang, H, Longley, MJ, Pan, H	Nucleic Acids Res	2018
33230761	In Vitro Analysis of mtDNA Replication Falkenberg, M, Uhler, JP	Methods Mol Biol	2021
20798345	G-quadruplex structures in RNA stimulate mitochondrial transcription termination and primer formation Falkenberg, M, Wanrooij, PH, Uhler, JP, Gustafsson, CM, Simonsson, T	Proc Natl Acad Sci U S A	2010
22207204	The interface of transcription and DNA replication in the mitochondria Kasiviswanathan, R, Copeland, WC, Collins, TR	Biochim Biophys Acta	2012
9182553	In vivo determination of replication origins of human mitochondrial DNA by ligation-mediated polymerase chain reaction Irie, T, Miyako, K, Takeshige, K, Kai, Y, Kang, D	J Biol Chem	1997
30239839	Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication Reyes, A, Cluett, TJ, Wood, SR, Holt, IJ, Spelbrink, JN, Spinazzola, A, Akman, G, Kazak, L, Mitchell, A	Nucleic Acids Res	2018
8670814	RNA-DNA hybrid formation at the human mitochondrial heavy-strand origin ceases at replication start sites: an implication for RNA-DNA hybrids serving as primers Clayton, DA, Xu, B	EMBO J	1996
29880722	Mitochondrial DNA replication in mammalian cells: overview of the pathway Falkenberg, M	Essays Biochem	2018
26303841	Primer removal during mammalian mitochondrial DNA replication Falkenberg, M, Uhler, JP	DNA Repair (Amst)	2015
29290614	Topoisomerase 3α Is Required for Decatenation and Segregation of Human mtDNA Taylor, RW, Gorman, GS, Larsson, NG, Chinnery, PF, Gustafsson, CM, Sommerville, EW, Basu, S, Hoberg, E, Falkenberg, M, Nadalutti, CA, Larsson, E, Turnbull, DM, Nicholls, TJ, Motori, E, Griffith, JD	Mol Cell	2018
25474639	In vivo occupancy of mitochondrial single-stranded DNA binding protein supports the strand displacement mode of DNA replication Sabouri, N, Falkenberg, M, Jemt, E, Zhu, X, Miralles Fusté, J, Shi, Y, Wanrooij, S, Gustafsson, CM, Persson, Ö	PLoS Genet	2014
23595151	Mitochondrial DNA replication proceeds via a 'bootlace' mechanism involving the incorporation of processed transcripts Reyes, A, Yasukawa, T, Wood, SR, Holt, IJ, Kazak, L, Jacobs, HT	Nucleic Acids Res	2013
12437923	Biased incorporation of ribonucleotides on the mitochondrial L-strand accounts for apparent strand-asymmetric DNA replication Gringeri, E, Reyes, A, Bowmaker, M, Angeli, P, Yang, MY, Holt, IJ, Vergani, L, Jacobs, HT	Cell	2002
32283748	TWINKLE and Other Human Mitochondrial DNA Helicases: Structure, Function and Disease Falkenberg, M, Peter, B	Genes (Basel)	2020
34423452	Mechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL Temiakov, D, Zamudio-Ochoa, A, Anikin, M, Sarfallah, A	EMBO J	2021
27220468	MGME1 processes flaps into ligatable nicks in concert with DNA polymerase γ during mtDNA replication Falkenberg, M, Milenkovic, D, Matic, S, Nicholls, TJ, Thörn, C, Uhler, JP, Gustafsson, CM	Nucleic Acids Res	2016
29931097	An overview of mammalian mitochondrial DNA replication mechanisms Yasukawa, T, Kang, D	J Biochem	2018
23090476	In vivo mutagenesis reveals that OriL is essential for mitochondrial DNA replication Falkenberg, M, Larsson, NG, Stewart, JB, Miralles Fusté, J, Wanrooij, PH, Samuelsson, T, Wanrooij, S, Gustafsson, CM	EMBO Rep	2012
16230534	Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism Cecconi, C, Bustamante, C, Tkachuk, AN, Clayton, DA, Brown, TA	Genes Dev	2005
22965135	A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop Falkenberg, M, Westerlund, F, Shi, Y, Wanrooij, PH, Uhler, JP, Gustafsson, CM	Nucleic Acids Res	2012
10677218	Human mitochondrial DNA polymerase holoenzyme: reconstitution and characterization Johnson, AA, Tsai, Yc, Graves, SW, Johnson, KA	Biochemistry	2000
36381197	Mitochondrial DNA: Consensuses and Controversies Alexeyev, M, Shokolenko, I	DNA (Basel)	2022
34500456	A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy Piccini, D, Giannattasio, M, Foiani, M, Kosar, M	Nucleic Acids Res	2021
2982153	Priming of human mitochondrial DNA replication occurs at the light-strand promoter Clayton, DA, Chang, DD	Proc Natl Acad Sci U S A	1985