Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication

Abstract Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication switches to a strand-coupled mechanism only where transcripts are scarce, thereby establishing a direct correlation between transcript availability and the mechanism of replication. Thus, these findings support a critical role of mitochondrial transcripts in the strand-asynchronous mechanism of mitochondrial DNA replication; and, as a corollary, mitochondrial RNA availability and RNA/DNA hybrid formation offer means of regulating the mechanisms of DNA replication in the organelle.

[1]  M. Gefter,et al.  DNA Replication , 2019, Advances in Experimental Medicine and Biology.

[2]  H. Jacobs,et al.  Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase , 2018, Nucleic acids research.

[3]  H. Masai,et al.  Potent DNA strand annealing activity associated with mouse Mcm2∼7 heterohexameric complex , 2017, Nucleic acids research.

[4]  M. Tigano,et al.  Single-Molecule Analysis of mtDNA Replication Uncovers the Basis of the Common Deletion. , 2017, Molecular cell.

[5]  H. Houlden,et al.  Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria , 2016, Proceedings of the National Academy of Sciences.

[6]  A. J. Bendich,et al.  DNA maintenance in plastids and mitochondria of plants , 2015, Front. Plant Sci..

[7]  H. Jacobs,et al.  Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication , 2015, Proceedings of the National Academy of Sciences.

[8]  M. Valle,et al.  The hexameric structure of the human mitochondrial replicative helicase Twinkle , 2015, Nucleic acids research.

[9]  H. Jacobs,et al.  A Rolling Circle Replication Mechanism Produces Multimeric Lariats of Mitochondrial DNA in Caenorhabditis elegans , 2015, PLoS genetics.

[10]  C. Gustafsson,et al.  In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication , 2014, PLoS genetics.

[11]  H. Jacobs,et al.  Unique features of DNA replication in mitochondria: A functional and evolutionary perspective , 2014, BioEssays : news and reviews in molecular, cellular and developmental biology.

[12]  F. Storici,et al.  Transcript RNA-templated DNA recombination and repair , 2014, Nature.

[13]  M. Minczuk,et al.  Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease , 2014, Human molecular genetics.

[14]  H. Jacobs,et al.  Mitochondrial DNA replication proceeds via a ‘bootlace’ mechanism involving the incorporation of processed transcripts , 2013, Nucleic acids research.

[15]  A. Reyes,et al.  Human mitochondrial DNA replication. , 2012, Cold Spring Harbor perspectives in biology.

[16]  J. Walker,et al.  Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis , 2012, Nucleic acids research.

[17]  Smita S. Patel,et al.  Human Mitochondrial DNA Helicase TWINKLE Is Both an Unwinding and Annealing Helicase* , 2012, The Journal of Biological Chemistry.

[18]  Hiroyuki Tsutsui,et al.  High mitochondrial DNA copy number has detrimental effects in mice. , 2010, Human molecular genetics.

[19]  H. Jacobs,et al.  Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid. , 2010, Journal of molecular biology.

[20]  H. Jacobs,et al.  Human Heart Mitochondrial DNA Is Organized in Complex Catenated Networks Containing Abundant Four-way Junctions and Replication Forks , 2009, The Journal of Biological Chemistry.

[21]  M. Falkenberg,et al.  The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities , 2007, Nucleic acids research.

[22]  J. N. Spelbrink,et al.  Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes , 2007, Nucleic acids research.

[23]  H. Jacobs,et al.  Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand , 2006, The EMBO journal.

[24]  T. Shutt,et al.  Twinkle, the Mitochondrial Replicative DNA Helicase, Is Widespread in the Eukaryotic Radiation and May Also Be the Mitochondrial DNA Primase in Most Eukaryotes , 2006, Journal of Molecular Evolution.

[25]  C. Richardson,et al.  Acidic Residues in the Nucleotide-binding Site of the Bacteriophage T7 DNA Primase* , 2005, Journal of Biological Chemistry.

[26]  H. Jacobs,et al.  A bidirectional origin of replication maps to the major noncoding region of human mitochondrial DNA. , 2005, Molecular cell.

[27]  A. Reyes,et al.  Bidirectional Replication Initiates at Sites Throughout the Mitochondrial Genome of Birds* , 2005, Journal of Biological Chemistry.

[28]  J. Fish,et al.  Discovery of a Major D-Loop Replication Origin Reveals Two Modes of Human mtDNA Synthesis , 2004, Science.

[29]  N. Ashley,et al.  Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number. , 2004, Human molecular genetics.

[30]  E. Schon,et al.  Two direct repeats cause most human mtDNA deletions. , 2004, Trends in genetics : TIG.

[31]  H. Jacobs,et al.  Mammalian Mitochondrial DNA Replicates Bidirectionally from an Initiation Zone* , 2003, Journal of Biological Chemistry.

[32]  P. Dijkwel,et al.  The Dihydrofolate Reductase Origin of Replication Does Not Contain Any Nonredundant Genetic Elements Required for Origin Activity , 2003, Molecular and Cellular Biology.

[33]  S. Gerbi,et al.  Developmental Changes in the Sciara II/9A Initiation Zone for DNA Replication , 2002, Molecular and Cellular Biology.

[34]  H. Jacobs,et al.  Biased Incorporation of Ribonucleotides on the Mitochondrial L-Strand Accounts for Apparent Strand-Asymmetric DNA Replication , 2002, Cell.

[35]  J. Diffley,et al.  MCM2-7 proteins are essential components of prereplicative complexes that accumulate cooperatively in the nucleus during G1-phase and are required to establish, but not maintain, the S-phase checkpoint. , 2001, Molecular biology of the cell.

[36]  G. Comi,et al.  Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria , 2001, Nature Genetics.

[37]  C. Newlon,et al.  Two Compound Replication Origins in Saccharomyces cerevisiae Contain Redundant Origin Recognition Complex Binding Sites , 2001, Molecular and Cellular Biology.

[38]  H. Jacobs,et al.  Coupled Leading- and Lagging-Strand Synthesis of Mammalian Mitochondrial DNA , 2000, Cell.

[39]  K. Takeshige,et al.  In Vivo Determination of Replication Origins of Human Mitochondrial DNA by Ligation-mediated Polymerase Chain Reaction* , 1997, The Journal of Biological Chemistry.

[40]  T. Kusakabe,et al.  Gene 4 DNA Primase of Bacteriophage T7 Mediates the Annealing and Extension of Ribo-oligonucleotides at Primase Recognition Sites* , 1997, The Journal of Biological Chemistry.

[41]  F. Studier,et al.  Biochemical Analysis of Mutant T7 Primase/Helicase Proteins Defective in DNA Binding, Nucleotide Hydrolysis, and the Coupling of Hydrolysis with DNA Unwinding* , 1996, The Journal of Biological Chemistry.

[42]  P. Dijkwel,et al.  Characterizing replication intermediates in the amplified CHO dihydrofolate reductase domain by two novel gel electrophoretic techniques , 1996, Molecular and cellular biology.

[43]  R. D. Little,et al.  Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin , 1995, Molecular and cellular biology.

[44]  D. Lilley,et al.  DNA replication, 2nd edn , 1992 .

[45]  P. Dijkwel,et al.  Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain , 1990, Cell.

[46]  W. L. Fangman,et al.  The localization of replication origins on ARS plasmids in S. cerevisiae , 1987, Cell.

[47]  D. A. Clayton,et al.  Replication of animal mitochondrial DNA , 1982, Cell.

[48]  F. Sanger,et al.  Sequence and organization of the human mitochondrial genome , 1981, Nature.

[49]  J. Posakony,et al.  Nucleotide sequence of a region of human mitochondrial DNA containing the precisely identified origin of replication , 1979, Nature.

[50]  A. Reyes,et al.  Analysis of replicating mitochondrial DNA by two-dimensional agarose gel electrophoresis. , 2007, Methods in molecular biology.

[51]  B. J. Brewer,et al.  Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. , 1995, Methods in enzymology.

[52]  D. Chang,et al.  Priming of human mitochondrial DNA replication occurs at the light-strand promoter. , 1985, Proceedings of the National Academy of Sciences of the United States of America.