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Methods in Molecular Biology 2615 Thomas Nicholls · Jay Uhler Maria Falkenberg Editors Mitochondrial DNA Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Mitochondrial DNA Methods and Protocols Edited by Thomas J. Nicholls Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK Jay P. Uhler Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden Maria Falkenberg Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden Editors ThomasJ.Nicholls JayP.Uhler WellcomeCentreforMitochondrial DepartmentofMedicalBiochemistryandCellBiology Research UniversityofGothenburg NewcastleUniversity Gothenburg,Sweden NewcastleuponTyne,UK MariaFalkenberg DepartmentofMedicalBiochemistry andCellBiology UniversityofGothenburg Gothenburg,Sweden ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2921-5 ISBN978-1-0716-2922-2 (eBook) https://doi.org/10.1007/978-1-0716-2922-2 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Mitochondria, the energy-generating and metabolic hubs of eukaryotic cells, possess their owngenomecalledmitochondrialDNAormtDNA.Mitochondrialgenomesvarywidelyin size and structure between kingdoms, from small and compact molecules in mammals to massive,complexmultimericgenomesinsomeplants,butareunitedbytheircentralrolein encoding components of the oxidative phosphorylation system. Human mtDNA was first discovered in the 1960s and is a multicopy genome found in the form of thousands of individual nucleoprotein complexes, called nucleoids, spread around the mitochondrial network.ThecopynumberofhumanmtDNAismaintainedbyaDNAreplicationmachin- ery of largely bacteriophage and bacterial origin, which operates independently of nuclear DNA replication and the cell cycle. Detailed genetic studies of mtDNA using classical molecular genetic techniques have long been hampered by the lack of a routine system to transform the mitochondria of human cells, although these systems exist for some model organisms, such as yeast. In vitro studies have also proven invaluable for the study of mitochondrialgeneticprocesses. An inability to maintain sufficient copies of mtDNA per cell, or the accumulation of mutationsordeletionsinasubsetofmtDNAmolecules,isacauseofmitochondrialdisease inhumans.Theserarebutdevastatingmetabolicdiseasescanmanifestatanystageoflifeand can result from defects arising in the mtDNA itself, or alternatively from pathological variants in the nuclear genes that encode the mtDNA replication and expression machineries. In this volume ofMethodsin MolecularBiology: MitochondrialDNA, now inits fourth edition,weaimtoprovideabroadrangeofprotocolsthatcanbeusedtostudymtDNAon molecular,cellular,andwholeorganismlevels,bothinvivoandinvitro.Thebookisdivided intosixparts.PartIprovidesmethodsfor theisolationofmitochondriaandmtDNAfrom differentspeciesandcelltypes,whichcanbeusedasastartingpointfor furtheranalysis.In Part II, protocols are presented for the visualization and quantification of mtDNA using differentmicroscopytechniques.PartIIIcontainsmethodsforidentifyingandcharacteriz- ing the mtDNA-interacting proteins of the mitochondrial nucleoid. Protocols for the analysis of mtDNA replication and repair are given in Part IV, utilizing reconstituted invitrosystemsaswellasusingmaterialisolatedfromcellsandtissues.Moderntechniques for the in vivo modification of mtDNA are presented in Part V. Finally, Part VI provides methodsfortheinvestigationofmtDNAinpathologicalstatesandforgeneticdiagnosticsin casesofhumanmitochondrialdisease. Wehopethatthesemethodswillbeusefulandinformativeforresearchersandclinicians from all career stages with an interest in mitochondrial DNA, from students to seasoned professors.Wearegratefultoalloftheauthorsthathavecontributedtothevolumefortheir hard work and for their willingness to share their experience and expertise with the wider scientificcommunity. NewcastleuponTyne,UK ThomasJ.Nicholls Gothenburg,Sweden JayP.Uhler Gothenburg,Sweden MariaFalkenberg v Contents Preface ..................................................................... v Contributors................................................................. xi PART I PURIFICATION METHODS FOR MITOCHONDRIAL DNA 1 IsolationofFunctionalMitochondriaandPuremtDNA fromMurineTissues ....... ........ ....... ....... ........ ....... ........ 3 DieuHienRozsivalova,MilicaPopovic,HarshitaKaul, andAleksandraTrifunovic 2 IsolatingMitochondria,Mitoplasts,andmtDNAfromCultured MammalianCells .......... ........ ....... ....... ........ ....... ........ 17 KatjaE.MengerandThomasJ.Nicholls 3 CouplingDifferentialCentrifugationwithExonucleaseTreatment andSizeExclusionChromatography(DIFSEC)forPurification ofmtDNAfromMammalianCells ........ .. ...... ....... .. ... .... ........ 31 AndrewM.ShawandPayamA.Gammage 4 IsolationandQualityControlofYeastMitochondria......... ....... ........ 41 AsliArasTaskin,DaianaNerinaMoretti, F.NoraVögtle,andChrisMeisinger 5 MitochondrialDNAIsolationfromPlants .......... ........ ....... ........ 57 Fre´de´riqueWeber-Lotfi,ArnaudFertet,RokasKubilinskas, andCle´mentineWallet,andJose´M.Gualberto PART II VISUALISING MTDNA 6 VisualizetheDistributionandDynamicsofMitochondrialDNA(mtDNA) NucleoidswithMultipleLabelingStrategies......... ........ ....... .... .... 79 XiangjunDi,JinshanQin,YujieSun,andQianPeterSu 7 VisualizationofmtDNAUsingFISH ....... ....... ........ ....... ........ 89 XieXieandXuefengZhu 8 InSituAnalysisofMitochondrialDNASynthesisUsingMetabolicLabeling CoupledtoFluorescenceMicroscopy........ ....... ........ ....... ........ 99 JohnA.SmolkaandSamanthaC.Lewis 9 MeasurementofNucleoidSizeUsingSTEDMicroscopy ..... ....... ........ 107 ElisaMotori PART III MITOCHONDRIAL DNA INTERACTING PROTEINS 10 HowtoQuantifyDNACompactionbyTFAMwithAcousticForce SpectroscopyandTotalInternalReflectionFluorescenceMicroscopy ......... 121 MartialMartucci,LouisDebar,SietvandenWildenberg, andGeraldineFarge vii viii Contents 11 AssessingTFAMBindingtoHumanMitochondrialDNA .... ....... ........ 139 Takehiro YasukawaandDongchonKang 12 IdentificationofProximityInteractorsofMammalianNucleoid ProteinsbyBioID ......... ........ ....... ....... ........ ....... ........ 153 MariJ.AaltonenandHanaAntonicka 13 LocalizationofMitochondrialNucleoidsbyTransmissionElectron MicroscopyUsingtheTransgenicExpressionoftheMitochondrial HelicaseTwinkleandAPEX2 ....... ....... ....... ........ ....... ........ 173 DavidPla-Martıń,FelixBabatz,andAstridC.Schauss PART IV MITOCHONDRIAL DNA REPLICATION AND REPAIR 14 InVitroAssaysofTWINKLEFunction ..... ....... ........ ....... ........ 191 JayP.Uhler,UlrikaAlexandersson,andMariaFalkenberg 15 RollingCircleReplicationandBypassofDamagedNucleotides ...... ........ 203 JosefinM.E.Forslund,GorazdStojkovicˇ,andSjoerdWanrooij 16 StudyingMitochondrialNucleicAcidSynthesisUtilizingIntact IsolatedMitochondria...... ........ ....... ....... ........ ....... ........ 219 JelenaMisicandDusankaMilenkovic 17 FunctionalAssessmentofMitochondrialDNAMaintenancebyDepletion andRepopulationUsing2’,3’-DideoxycytidineinCulturedCells..... ....... . 229 Ga´borZsurka,GenevieveTrombly,SusanneSchöler, DanielBlei,andWolframS.Kunz 18 AnalysisofMitochondrialDNAReplicationbyTwo-Dimensional AgaroseGelElectrophoresis......... ....... ....... ........ ....... ........ 241 SteffiGoffartandJaakkoPohjoisma€ki 19 QuantitativeAnalysisofNucleosideTriphosphatePoolsinMouse MuscleUsingHydrophilicInteractionLiquidChromatographyCoupled withTandemMassSpectrometryDetection......... ........ ....... ........ 267 SushmaSharma,ZiqingKong,ShaodongJia,PhongTran, AnnaKarinNilsson,andAndreiChabes 20 DetectionofUV-InducedDeletionsinMitochondrialDNA......... ........ 281 GabrieleA.FontanaandHaileyL.Gahlon 21 DeterminationoftheRibonucleotideContentofmtDNA UsingAlkalineGels ........ ...... .. .... ... ....... ........ ....... ........ 293 ChocoMichaelGorospe,BrunoMarçalRepoleˆs, andPaulinaH.Wanrooij 22 5′-EndMappinginHumanMitochondrialDNA .... ........ ....... ........ 315 AndranikDurgaryanandAndersR.Clausen PART V MODIFYING MITOCHONDRIAL DNA 23 ManipulationofMurineMitochondrialDNAHeteroplasmy withmtZFNs....... ....... ........ ....... ....... ........ ....... ..... ... 329 PavelA.NashandMichalMinczuk Contents ix 24 BiolisticTransformationofChlamydomonasreinhardtii andSaccharomycescerevisiaeMitochondria .......... ........ ....... ........ 345 NathalieBonnefoyandClaireRemacle 25 AMethodforPreciselyIdentifyingModificationstoPlantMitochondrial GenomesbymitoTALENs.......... ....... ....... ........ ....... ........ 365 TomohikoKazamaandShin-ichiArimura PART VI MITOCHONDRIAL DNA IN HUMAN DISEASE 26 MitochondrialDNASequencingandHeteroplasmyQuantification byNextGenerationSequencing..... ....... ....... ........ ....... ... ..... 381 AndreaLegati,DanieleGhezzi,andCarloViscomi 27 GenomicStrategiesinMitochondrialDiagnostics.... ........ ....... ........ 397 DashaDeen,CharlotteL.Alston,GavinHudson, RobertW.Taylor,andAngelaPyle 28 MitochondrialDNAEnrichmentforSensitiveNext-Generation Sequencing ........ ....... ........ ....... ....... ........ ....... ........ 427 ShilanWu,MatthewJ.Longley,ScottA.Lujan, ThomasA.Kunkel,andWilliamC.Copeland 29 SingleCellAnalysisofMitochondrialDNADeletions........ ....... ........ 443 HelenA.L.Tuppen,AmyK.Reeve,andAmyE.Vincent Index .......... ........ ....... ........ ....... ....... ........ ....... ........ 465 Contributors MARIJ.AALTONEN • MontrealNeurologicalInstitute,McGillUniversity,Montreal,QC, Canada;DepartmentofHumanGenetics,McGillUniversity,Montreal,QC,Canada ULRIKAALEXANDERSSON • DepartmentofMedicalBiochemistryandCellBiology,University ofGothenburg,Gothenburg,Sweden CHARLOTTEL.ALSTON • WellcomeCentreforMitochondrialResearch,Translationaland ClinicalResearchInstitute,FacultyofMedicalSciences,NewcastleUniversity,Newcastle uponTyne,UK;NHSHighlySpecialisedServicesforRareMitochondrialDisorders,Royal VictoriaInfirmary,NewcastleuponTyneHospitalsNHSFoundationTrust,Newcastle uponTyne,UK HANAANTONICKA • MontrealNeurologicalInstitute,McGillUniversity,Montreal,QC, Canada;DepartmentofHumanGenetics,McGillUniversity,Montreal,QC,Canada SHIN-ICHIARIMURA • GraduateSchoolofAgricultureandLifeSciences,UniversityofTokyo, Tokyo,Japan FELIXBABATZ • CologneClusterofExcellenceonCellularstressresponseinAging-associated Disease,CECAD,UniversityofCologne,Cologne,Germany DANIELBLEI • DivisionofNeurochemistry,InstituteofExperimentalEpileptologyand CognitionResearch,UniversityofBonn,Bonn,Germany NATHALIEBONNEFOY • InstituteofIntegrativeBiologyoftheCell(I2BC),CEA,CNRS, UnivParis-Sud,Universite´Paris-Saclay,Gif-sur-Yvettecedex,France ANDREICHABES • DepartmentofMedicalBiochemistryandBiophysics,Umea˚ University, Umea˚,Sweden ANDERSR.CLAUSEN • InstituteofBiomedicine,UniversityofGothenburg,Gothenburg, Sweden WILLIAMC.COPELAND • GenomeIntegrityandStructuralBiologyLaboratory, MitochondrialDNAReplicationGroup,NationalInstituteofEnvironmentalHealth Sciences,NationalInstitutesofHealth,ResearchTrianglePark,NC,USA LOUISDEBAR • UniversiteĆlermontAuvergne,CNRS,LaboratoiredePhysiquede Clermont,Clermont-Ferrand,France DASHADEEN • WellcomeCentreforMitochondrialResearch,TranslationalandClinical ResearchInstitute,FacultyofMedicalSciences,NewcastleUniversity,NewcastleuponTyne, UK XIANGJUNDI • SchoolofBiomedicalEngineering,FacultyofEngineeringandInformation Technology,UniversityofTechnologySydney,Sydney,NSW,Australia ANDRANIKDURGARYAN • InstituteofBiomedicine,UniversityofGothenburg,Gothenburg, Sweden MARIAFALKENBERG • DepartmentofMedicalBiochemistryandCellBiology,Universityof Gothenburg,Gothenburg,Sweden GERALDINEFARGE • UniversiteĆlermontAuvergne,CNRS,LaboratoiredePhysiquede Clermont,Clermont-Ferrand,France ARNAUDFERTET • InstitutdeBiologieMolećulairedesPlantes,CNRS,Universite´de Strasbourg,Strasbourg,France GABRIELEA.FONTANA • DepartmentofHealthSciencesandTechnology,ETHZurich, Zurich,Switzerland xi