title: TWINKLE is an essential mitochondrial helicase required for synthesis of nascent D-loop strands and complete mtDNA replication.
abstract: Replication of the mammalian mitochondrial DNA (mtDNA) is dependent on the minimal replisome, consisting of the heterotrimeric mtDNA polymerase (POLG), the hexameric DNA helicase TWINKLE and the tetrameric single-stranded DNA-binding protein (mtSSB). TWINKLE has been shown to unwind DNA during the replication process and many disease-causing mutations have been mapped to its gene. Patients carrying Twinkle mutations develop multiple deletions of mtDNA, deficient respiratory chain function and neuromuscular symptoms. Despite its importance in human disease, it has been unclear whether TWINKLE is the only replicative DNA helicase in mammalian mitochondria. Furthermore, a substantial portion of mtDNA replication events is prematurely terminated at the end of mitochondrial control region (D-loop) and it is unknown whether TWINKLE also has a role in this abortive replication. Here, we present a conditional mouse knockout for Twinkle and demonstrate that TWINKLE is essential for mouse embryonic development and thus is the only replicative DNA helicase in mammalian mitochondria. Conditional knockout of Twinkle results in severe and rapid mtDNA depletion in heart and skeletal muscle. No replication intermediates or deleted mtDNA molecules are observed after Twinkle knockout, suggesting that TWINKLE once loaded is very processive. We also demonstrate that TWINKLE is essential for nascent H-strand synthesis in the D-loop, thus showing that there is no separate DNA helicase responsible for replication of this region. Our data thus suggest that the relative levels of abortive D-loop synthesis versus complete mtDNA replication are regulated and may provide a mechanism to control progression to complete mtDNA replication.
authors: Max Planck Institute for Biology of Ageing, Robert-Koch-Strasse 21, Cologne, Germany.
authors: Dusanka
authors: D
authors: Milenkovic
authors: Stanka
authors: S
authors: Matic
authors: Inge
authors: I
authors: Kühl
authors: Benedetta
authors: B
authors: Ruzzenente
authors: Christoph
authors: C
authors: Freyer
authors: Elisabeth
authors: E
authors: Jemt
authors: Chan Bae
authors: CB
authors: Park
authors: Maria
authors: M
authors: Falkenberg
authors: Nils-Göran
authors: NG
authors: Larsson
mesh_terms: D000818:Animals; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D004272:DNA, Mitochondrial / Q000096:biosynthesis* / Q000235:genetics; D030342:Genetic Diseases, Inborn / Q000201:enzymology / Q000235:genetics; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009468:Neuromuscular Diseases / Q000201:enzymology / Q000235:genetics
chemical_list: D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; C513299:Twnk protein, mouse; D004265:DNA Helicases; C431576:TWNK protein, human
references: J Cell Biol. 2011 May 30;193(5):809-18
references: 21606204
references: J Mol Biol. 2008 Mar 28;377(3):691-705
references: 18279890
references: J Biol Chem. 2007 Mar 30;282(13):9436-9444
references: 17272269
references: Hum Mol Genet. 2005 Jul 1;14(13):1775-83
references: 15888483
references: Nat Genet. 2001 Jul;28(3):223-31
references: 11431692
references: Nucleic Acids Res. 2010 Oct;38(19):6577-88
references: 20566479
references: J Biol Chem. 2003 Dec 5;278(49):48627-32
references: 12975372
references: Nucleic Acids Res. 2007;35(10):3238-51
references: 17452351
references: Nucleic Acids Res. 2005 Jul 08;33(12):3812-20
references: 16006625
references: Proc Natl Acad Sci U S A. 1981 Oct;78(10):6116-20
references: 6273850
references: Nucleic Acids Res. 2011 Nov;39(21):9238-49
references: 21840902
references: Cell. 1982 Apr;28(4):693-705
references: 6178513
references: PLoS One. 2012;7(8):e43954
references: 22952820
references: J Mol Evol. 2006 May;62(5):588-99
references: 16612544
references: Hum Mol Genet. 2004 Dec 15;13(24):3219-27
references: 15509589
references: Proc Natl Acad Sci U S A. 2011 Aug 16;108(33):13534-9
references: 21808029
references: J Mol Biol. 2010 Apr 16;397(5):1144-55
references: 20184890
references: Anal Biochem. 1991 Dec;199(2):223-31
references: 1812789
references: Biochim Biophys Acta. 1971 Nov 29;254(1):1-14
references: 4943979
references: Cell Metab. 2011 May 4;13(5):527-39
references: 21531335
references: J Biol Chem. 2008 Aug 29;283(35):23964-71
references: 18593709
references: Cell Metab. 2009 Apr;9(4):386-97
references: 19356719
references: Mitochondrion. 2007 Sep;7(5):311-21
references: 17698423
references: Methods Mol Biol. 2009;554:103-19
references: 19513670
references: EMBO J. 2004 Jun 16;23(12):2423-9
references: 15167897
references: Hum Mol Genet. 2009 Jan 15;18(2):328-40
references: 18971204
references: Annu Rev Biochem. 2007;76:679-99
references: 17408359
references: EMBO Rep. 2012 Dec;13(12):1130-7
references: 23090476
references: Genomics. 2001 Apr 1;73(1):56-65
references: 11352566
references: Mol Cell. 2009 Aug 28;35(4):490-501
references: 19646907
references: Cell. 2000 Mar 3;100(5):515-24
references: 10721989
references: Mol Biol Cell. 2007 Sep;18(9):3225-36
references: 17581862
references: Biochim Biophys Acta. 2010 Aug;1797(8):1378-88
references: 20417176
references: Mol Biol Cell. 2010 Apr 15;21(8):1315-23
references: 20200222
references: Nat Genet. 1999 Jan;21(1):133-7
references: 9916807
references: Nat Struct Mol Biol. 2011 Oct 30;18(11):1290-6
references: 22037171
references: Cell. 2007 Jul 27;130(2):273-85
references: 17662942
references: Neurology. 1997 May;48(5):1244-53
references: 9153451
references: Hum Mol Genet. 2004 May 1;13(9):935-44
references: 15016765
references: Biochem Biophys Res Commun. 1994 May 16;200(3):1374-81
references: 8185589
references: Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11122-7
references: 18685103
references: Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17687-92
references: 16301523
references: Cell. 2012 Mar 16;148(6):1145-59
references: 22424226
references: Hum Mol Genet. 2010 Jul 1;19(13):2695-705
references: 20413656
references: Nat Genet. 1998 Mar;18(3):231-6
references: 9500544
references: Mol Cell. 2010 Jan 15;37(1):67-78
references: 20129056
references: Annu Rev Biochem. 2010;79:683-706
references: 20350166
references: PLoS Genet. 2011 Oct;7(10):e1002324
references: 22022283
references: EMBO J. 2006 Nov 15;25(22):5358-71
references: 17066082
references: EMBO J. 2012 Jan 18;31(2):443-56
references: 22045337