医学遗传与胚胎发育 Chapter 3 DNA Replication and Repair

DNAOrganization,Replication,andRepair12Nucleoside:GlycosylatednucleobaseNucleotide:PhosphateestersofnucleosidesChemicalNatureofDNA3Nucleobases4ModifiedNucleobasesModifiedbasesarealsofoundinbothDNAandRNA,althoughthesemodificationsoccuronlyafterDNAsynthesishasbeencompleted.Polynucleotidechains:Nucleotidesarepolymerizeddirectionallythroughthe3'hydroxylofthedeoxyribose.DNA:Structures5A=T;G=C.Therefore,thebasesfoundinoneDNAstrand(ortemplate)determinetheorderofthecomplimentarystrand.Thissuggestsacopyingmechanismforthetransmissionofgeneticinformation.Double-HelicalDNA6DNAfunction:

Avery’sexperimentDNAbasecomposition:Chargaff'srule(theconcentrationofAequalstheconcentrationofT;andtheconcentrationofGequalstheconcentrationofC)theconceptofcomplimentarystrandsevolved.DNAX-raydiffractionpatternconfirmeddouble-strandedhelicalstructure.Double-HelicalDNA7Double-HelicalDNAComplementary&Anti-parallel89FactorsthatStabilizeDouble-HelicalDNA.1. BaseStacking.Hydrophobicbasesresideintheinteriorofthehelixwherewaterisexcluded,therebypermittinghydrophobicbasestackinginteractions(vanderWaalsforces).Thisisthemajorentropiccontributiontohelixstabilization.Thehydrophilicphosphatebackboneissolvatedbywater.2. BasePairing.TheA-Tbasepairoccurswith2hydrogenbonds,whereastheG-Cbasepairoccurswith3hydrogenbonds.Therefore,theG-Cbasepairrequireshighertemperatureto"melt"thananA-Tbasepair.However,basepairingdoesnotcontributesubstantiallytothefreeenergyofhelixstabilization,butdoesprovidedirectionality,stabilityandspecificitytodoublehelixformation.10ThepredominantformofDNAistheB-conformation,however,otherconformationsalsoexistinnature,includingA-DNAandZ-DNA.Theseconformationsaffectthesizeanddepthofthemajorandminorgrovesofthedoublehelix.ConformationsofDouble-HelicalDNA11FeaturesofA,BandZDNA(seetable2.3) A-DNA B-DNA Z-DNA__Helixrotation Right-HandedRightHanded LeftHandedBasesperturn 10.7 9.7 12Helixriseperbasepair 2.6Å 3.4Å 3.7ÅBasetilt 20 6

71213NoncanonicalDNAStructuresSomeefforthasbeenmadetoextractDNAfromfossilizeddinosaurbonesthathavebeenburiedformanymillionsofyearsinaridclimates.InwhatformisthisDNAmostlikelytobefound? A.A B.B C.Z D.Tetraplex E.Completelydenatured14TheDNAmoleculeinpurewater: A:isinsoluble B:isacidic C:isbasic D:isviscous E:tastessalty15DNAPackagingChromatinDNAispackagedinthenucleusaschromatin,aDNA-proteincomplex.Chromatinthatistranscribedisreferredtoaseuchromatin,whileheterochromatinisnottranscribed1617 HistonesaresmallbasicproteinsthatparticipateinpackagingDNAbytheformationofnucleosomes.

Anucleosomecorecontainstwoeachofhistones2A,2B,3and4withDNAwrappedaroundthehistones.

Histone1occupiesthelinkerregionbetweennucleosomes.

Histonesarehighlyconservedproteins,whichattachtoDNAbyhighlyspecificsaltbridges.

HistonesandNucleosomes18Thenucleosomeconsistsofapproximately146bpofDNAcorrespondingto13/4superhelicalturnswoundaroundahistoneoctamer.TheH1histoneisassociatedwiththelinkerDNA1920ChangesinDNApackingandgeneexpressionoccurinpartbycovalentmodificationofcorehistones(H2A,H2B,H3,H4).Histonesundergoreversibleacetylationoflysine,catalyzedbytheenzymeshistoneacetylaseandhistonedeacetylase.Histometaillysinescanalsobemodifiedbymethylation,ADP-ribosylation,phosphorylation,glycosylation,ubiquitinationandsumoylation.

Epigenetics:HistoneCode21Nucleosomescanformhigherorderstructuressuchas30nmfibersresultinginfurthercompactingofDNA22DNAReplication A.Initiation B.Elongation C.Termination

ReplicationrequireshighfidelitybecausemistakesarememorizedComparetoRNA,ProteinSynthesis,ComparetoHIV23Intheory,DNAreplicationcanoccurbyadispersive,conservativeorsemiconservativemethod.Semi-conservativeSynthesisInreality,replicationoccursonlybyasemi-conservativemode.SynthesisrequiresaDNAtemplatefortheproductionofadaughterstrandfromtheold,orparentalstrand.24

TheMeselson-Stahlexperiment,195725ChemistryofChainElongationTransesterification26DNAsynthesisisinthe5'->3'directionatthemolecularlevel.DNApolymerasecleavespyrophosphatefromadNTPasitformsaphosphodiesterbondbetweenthe3'OHoftheprimerandthe5'Pofthesubstrate.Sincethenucleotideisaddedtothe3'endoftheprimer,synthesisisalways5'->3‘.ChemistryofChainElongation27DNAPolymerasesLarge(Klenow)fragmentofDNApolymeraseI.28DNAreplicationrequiresprimerstogetstartedandneedstemplatetoproceedDNApolymeraseisunabletopolymerizetwomononucleotides,itcanonlyaddnucleotidestoanexistingpolynucleotidestrand.AnenzymetermedprimaseformsashortsegmentofRNA,whichactsasaprimerforDNAsynthesis.Incontrasttothesynthesisofmostothermacromolecules,thesequenceofnucleicacidsisdeterminedbyatemplate.Thetemplatestranddirectssynthesis,thatis,itcontainstheinformationthatdeterminesthesequenceofthenewDNAstrands.2930Proofreadingiscarriedoutby3’→5’exonuclease3132DirectionofDNASynthesisDNAisreplicated5'-->3'inareplicationfork -leadingstrandisreplicated5'-->3' -laggingstrandmovesdiscontinuously3'-->5DNAsynthesisissemi-discontinuous33Continuousvs.discontinuoussynthesis.Incontinuoussynthesis,primaseformsanRNAsegmentwhichactsasaprimerforDNApolymeraseIII(PolIII)andtheleadingstrandissynthesizedwithoutinterruption.Indiscontinuoussynthesis,primaseformsanRNAsegmentandPolIIIaddsabout1000nucleotides,whichrepresentsanOkazakifragment.DNApolymeraseI(PolI)excisestheRNAwithits5'->3'exonucleaseactivityandreplacesitwithDNA.AfterPolIhasacted,the3'endofthelastnucleotidemustbeligatedtothe5'PofthesegmentmadebyPolIII.DNAligasecarriesoutthelatteractivity.34Thenewprimerelongated,Theoldprimerremoved,ThegapfilledThenicksealed35Firststep:separationofDNAdoublehelix36UntwistingParentalStrandsAsreplicationcontinues,strandseparationcausessupercoiling(tension)intheregionaheadofthegrowingfork.Thisispreventedbytheactionofatopoisomerases,whichnicktheDNAtoallowthestrandstountwistandthensealsthenick.Inprokaryotes,typeItopoisomerasesremovesonlynegativesupercoil.TypeIItopoisomerases,alsoreferredtoasDNAgyrase,whichisapowerswivelthatremovespositivesupercoilsinducesnegativesupercoils.37Topoisomerasemechanism:reversibletransesterification38Protein Role Clamp–loadingprotein Scaffoldthatbindspolymeraseshelicase SeparatesDNAstrandsandenablesprimasetoinitiatePrimase SynthesizesRNAprimers Topoisomerases UnwindsthedoublehelixremovingnegativesupercoilsSS-bindingproteins Stabilizessingle-strandedregionsDNAgyrase introducesnegativesuperhelicaltwistsDNApolymeraseIII SynthesizesDNADNApolymeraseI Erasesprimerandfillsgaps DNAligase JoinstheendsofDNA DNAmethylase adenosinemethylationmemorizesthereplicationDNAReplicaseSystem:Replisome39ProcaryoticDNAReplication40Replisome:bybindingtwopolymerasestogether,andloopingthediscontinuousstrand,DNAfeedsthroughthecomplex,rotatingasitpassesthrough41Bacterialpolymerases:PolI Fillsingapsinlaggingstrand;repairs,primerremovalPolII DNArepair,damagebypassPolIII PrimaryDNApolymerase,repairPolIV DamagebypassPolV DamagebypassPolI,IIandIIIhave3'-->5'exonucleaseactivity.PolIhas5'-->3'exonucleaseactivity(forprimerremoval)Eukaryoticpolymerases- primingactivityβ- repair,gapfiller,lowfidelity(lacks3’->5’exonucleaseactivity)- mitochondrialpolymerase- primarypolymerase- repairpolymerase42TerminationofReplicationinLinearGenomes:TelomeresInthegrowingreplicatingforkofalinearDNAmolecule,onestrandcangrowcontinuouslytotheend(the5'to3'orleadingstrand).However,thecomplementarytemplatestrandwiththelaggingdaughterstrandcannotbecompletelysynthesizedtotheend.Repetitivetelomeric(end)sequencesareaddedonbytelomerasetosolveaproblem.43TelomeraseisaribonucleoproteincomplexwithashortRNAstrandasanintegralpart;itcatalyzestheadditionofnew6-nttelomererepeatstothe3’endofaDNAchain.TelomeraseRNApartiallybase-pairwiththetelomericrepeatandservesasthetemplateforthereaction,whiletheproteincomponentfunctionsasareversetranscriptase,synthesizingDNAusingRNAtemplate.Aftera6-ntrepeatisadded,theenzymecandissociateandbindagain,andaddadditional6-ntrepeats.44ReverseTranscription-RNAsometimesservesasthetemplateinDNAsynthesis.Forinstance,retrovirusessynthesizeDNAbyareactioncatalyzedbyreversetranscriptaseinwhichRNAservesasthetemplate.ThegenomeofretrovirusesconsistsoftwoidenticalstrandsofRNA.45DNASynthesisinSPhaseDNAreplicationoccursonlyduringtheS(synthetic)phaseofthecellcycle.TheSphaseisusuallythelongestperiodofthecellcycle.46WhattypeofbondmustbemadebetweenOkazakifragmentsinordertomakeacompleteDNAstrand? A.Hydrogen B.Disulfide C.Ester D.Phosphodiester E.Ionic

47DNARecombination AgeneralphenomenonduringwhichtwostrandsofDNAmoleculesaresplicedtogethergivingrisetoanewDNAmoleculethatcontainsinformationfromeachstrand.It’sacontrolled“mixing”ofDNAthatincreasesgeneticdiversity.ThecontroloccursasonlysuitableorcloselyrelatedDNAmoleculescanrecombine.DNArecombinationandmutationarethetwoprincipalapproachesbywhichcellscreatevariationthatisrequiredformolecularevolutiontooccur.

Examples:•Chromosomal“crossingover”duringmeiosis.•IntegrationofviralDNAintoahost•Transductionof“hostcell”DNAintoarecipientcell.48Typesofrecombination:

Type Sequence Heterduplex Proteins DNA Homology sequences involved Synthesis__________________________________________________________________________Homologous Extensiveonboth long RecA,RecBCD,RuvC Some strands;notsequence specificSite-Specific Short, short Recombinases Some specificDNA sequenceonbothstrandsTranspositional Homologynotrequirednone Transposases Minor specificDNAsequenceis (onlytofillgaps) requiredononestrandNHEJ NONE none DNA-PK Minor (onlytofillgaps)

49Recombinationoccursbetweentwohomologous(largelycomplementary)slightlyvariantcopiesofasamechromosomespanningseveralthousandbases.HomologousRecombinationTherehasbeenanequal,reciprocalexchangebetweenthem.50Featuresofholidaymodel:HomologySymmetryofbothbreaksandstrandinvasionFour-stranded“Holidayjunction”asakeyintermediate.HomologousalignmentReciprocalinvasionBranchmigrationHolidayjunctionThegenearrangementdoesnotchange51ItcontributestotherepairofseveraltypesofDNAdamage,e.g.,stalledreplicationforks.Itensuresaccuratechromosomalsegregationduringmeiosis.Itcreatesgeneticdiversity.

HomologousRecombination52Site-SpecificRecombination:Occursatspecificsitesonbothrecombinationsites(20-200bp)withlimitedcomplementarysequences.SitespecificrecombinationisenzymemediatedandrequiresaconsensusDNAsequencetobindaproteinwithtypeItopoisomeraseactivityknownasarecombinase.Unlikehomologousrecombination,therelativepositionofnucleotidesandtheirsequenceisimportantasthepairingreactionisenzymemediated(extensivehomologyisnotrequired)andtheprocessisreversible.53Site-SpecificRecombinationThemostcommonexampleistherecombinationofbacteriophagelambdaandtheE.colichromosome;bothsiteshaveacommonsequenceofonly20nucleotides.54EffectsofSite-SpecificRecombination55III.TranspositionalRecombination:

TransposableelementsofDNA,orjumpinggenesortransposonscanmoveandintegratewithinorbetweenchromosomes.Itdiffersfromsite-specificrecombinationinthatspecificDNAsequencesarenotrequiredinthe“targetchromosome”,onlyinthedonorortransposon(15-25bp).Transpositioniscatalyzedbytransposases.(1)ThemostcommonareLINEs(longinterspersedelements;50,000perperhumangenome)andSINEs(shortinterspersedelementsorAluelements:500,000perhumangenome).(2)Thetransposoncanalsoencodeanentiregeneresultingingeneduplication.(3)Itcanintegratewithinagenetherebyinhibitingitsexpression.(4)Thetransposoncouldbeanenhancerelement,suchasc-myc,andintegrateupstreamofageneandchangeitsexpression.(5)Thespreadofantibiotic-resistanceelements.56TranspositionalRecombination57ClassI:Retrotransposons–copyandpaste(replicativetransposition)ClassII:DNAtransposons–cutandpaste(directtransposition)58MechanismofimmunoglobulingenerearrangementcatalyzedbyRAG1/RAG2.AprogrammedrecombinationreactionrelatedtotranspositionsjoinsimmunoglobulingenesegmentstoformmatureimmunoglobulingenesduringB-celldifferentiation.59DNADamageandMutationsMutationisdefinedasaninheritablechangeinachromosome.Afterreplicationthealteredbaseispermanentlyincorporatedintothegenome.

Therearetwotypesofmutations:1.Basesubstitutions–pointmutations substitutionofonebaseforanother:

-Missensemutation:changeanaminoacidcodonandproteinsequence.-Synonymousmutation

:doesnotaffectaminoacidcodonorproteinsequence.-Nonsensemutation

:changeanaminoacidcodontoastopcodon.

2.Frameshiftmutations Changeoftranslationalreadingframe.6061DeaminationDepurinationMethylationCross-linkingAlkylation62ThetwomostcommonDNAmutationsaredepurinationandcytosinedeamination.Depurinationoccursatarateof5000/day/genomeduetothermaldisruption;C-Udeaminationoccursatarateof100changes/day/genome.DeaminationconvertsaG-CbasepairintoapotentialA-Tbasepair.63CarcinogenslikeEthidiumBromidecancausemutationsincludingbaseinsertions.Intercalatingagent:mutagen&carcinogen64InmammalsthereisastrongcorrelationbetweentheaccumulationofmutationsandcancerInXerodermaPigmentosum,exposuretosunlightcausessevereskinreactionseventuallyresultinginskincancer.Incontrasttonormalcells,skincellsfromtheseindividualsareunabletoefficientlycarryoutexcisionrepairofDNAdamagedbyultravioletlight.DNArepairispossiblelargelybecausetheDNAmoleculeconsistsoftwocomplementarystrands.65DNARepairTherearetwomajormodesofexcisionrepair: baseexcisionrepair(BER) nucleotideexcisionrepair(NER)Basicsteps:RecognizedamageRemovedamagetoleaveagapResynthesizetofillthegapligatetorestorcontinuityofDNAbackboneBERGlycosylaseremovesbaseAPendonucleasecutsbackboneAPlyaseremovessugarDNApolymerasefillsgapDNAligasesealsnickNERDoubleexcisiondamageaspartofanoligonucleotideDNApolymerasefillsgapDNAligasesealsnic