DNA的生物合成和损伤修复DNA-Biosynthesis-and-DNA-Damage-Repa

Chapter15DNABiosynthesisandDNADamageRepairSectionOneTheGeneralFeaturesofReplicationofChromosomalDNA1.DNAreplicatessemiconservatively.⑴.WatsonandCrickpredictedthatDNAmightsemiconservativelyreplicate.thehypothesis___________p___________d___________p___________p____________d____________p⑵.In1958MeselsonandStahldemons-tratedthesemiconservativenatureofDNAreplicationinEcoli.Theexperiment:CsClequilibriumgradientdensityultracentri-fugationof15NlabeledEcoliDNA.ThedensityofDNAwasincreasedbylabelingitwith15N,aheavyisotopeofnitrogen.ThiswasdonebygrowingEcoli15ge-nerationsinamediumthatcontained15NH4Clasitsonlynitrogensource.15NDNAwasextractedandsubjectedtoCsClequilibriumgradientdensityultracen-trifugation.TheDNAbandpositionwasrecorded.Therewasonebandof15NDNA.Thebacteriaweretransferredtoan14NH4Clmediumandgrownforonegeneration.TheDNAdensitywasdeterminedagain.ThepositionoftheDNAbandwascomparedwiththatofthe15NDNA.Whatwastheresult?TherewasoneDNAband.Ithadalowerdensitythan15NDNAbecauseitspositionwasaboveonthatof15NDNA.Itwas15N/14NhybridDNA.Afteranothergenerationgrowinginthe14NH4ClmediumthebacterialDNAdensitywasdetermined.ThereweretwoDNAbands.OnehalfoftheDNAwas14NDNA,andanotherhalfwashybridDNA.Insucceedinggenerationstheratioof14NDNAtohybridDNAincreasedgradually.ThehybridDNAbecamelessandless..summaryDNAreplicatesinasemiconservativeman-ner.Whenthetwoparentalstrandssepa-rate,eachservesasthetemplateformakinganew,complementarystrand.2.ThepointatwhichseparationofthestrandsandsynthesisofnewDNAtakesplaceisknownasthereplicationfork.ThereplicationforkisY-shaped.Twoarms(V)areseparatedstrandswhichactasthetemplateandDNAsynthesisisactivelytakingplace.Thebody(I)istheparentalDNA.3.DNAreplicationisusuallybidirectional.⑴.Replicon:Anypiecewhichreplicatesasasingleunitiscalledareplicon.AllbacterialchromosomesandmanyphageandvirusDNAmoleculesarecircularandcomprisesinglereplicons.Incontrasteukaryoticchromosomesconsistofmultiplereplicons.⑵.Origin:Theinitiationwithinarepliconalwaysoccursatafixedpointknownastheorigin.⑶.Terminus:Inacircularrepliconthereisasingleterminationsiteroughly180°oppositetheuniqueorigin.SummaryInacircularrepliconreplicationbeginsfromthefixedoriginandformstworepli-cationforks.Thetworeplicationforksproceedbidirec-tionallyawayfromtheoriginandthestrandsarecopiedastheyseparateuntiltheterminusisreached.4.DNAreplicationissemidiscontinuous.⑴.ThemechanismofDNAreplicationallowsonlyforsynthesisina5’3’direction.⑵.ThetwostrandsofDNAareantiparallel.QuestionHowistheparentalstrandthatruns5’3’pastthereplicationforkcopied?Theanswerissemidiscontinuousreplica-tion.Ateachreplicationforkonestrand(thelead-ingstrand),whosetemplateruns3’5’pastthereplicationfork,issynthesizedasonecon-tinuouspiece,whiletheotherstrand(thelag-gingstrand),whosetemplateruns5’3’pastthereplicationforkismadediscontinuouslyasshortfragmentsinthereversedirection.TheseshortfragmentsarecalledOkazakifragments.TheyarejoinedbyDNAligaseandformthelaggingstrand.5.OriginscontainshortAT-richrepeatse-quences.⑴.Prokaryoticandeukaryoticoriginshavecommonfeatures:a.Theyconsistofmultipleuniqueshortrepeatsequences.b.Thesesequencesarerecognitionandbindingsitesofmulti-subunitinitiationfactors.c.ThesesequencesareusuallyAT-rich.⑵.Ecoli‘soriginiscalledoriC.Itis254bplongandcontainsthree13-bpdirectrepeatsandfour9-bpinvertedre-peats.6.DNAreplicationneedspriming.⑴.DNApolymerasescannotinitiateDNAreplicationbystartinganewDNAchain.Theycanonlyaddnucleotidestothe3’endofanexistentpieceofRNAorDNAunderthedirectionofthetemplate.TheexistentpieceofRNAorDNAarecalledprimer.⑵.TheleadingstrandandallOkazakifrag-mentsareprimedbysynthesisofashortpieceofRNA(anRNAprimer),whichisthenelongatedwithDNAbyDNApoly-merase.⑶.TherearealsoDNAprimingornucleotidepriming.7.Multi-enzymesandproteinsparticipateinDNAreplication.⑴.TopoisomerasesregulatethetypeandlevelofsupercoilingofdsDNA.⑵.HelicasesunwindthedsDNA.⑶.SSBsbindandstabilizethesingleDNAstrand.⑷.PrimasesynthesizestheRNAprimer.⑸.DNApolymeraseselongateDNAchains.⑹.DNAligasejoinsOkazakifragments.8.DNAreplicationisofhighfidelity.⑴.Therearetwotypesofreplicationerrors.a.base(nucleotide)substitution.b.nucleotideinsertionordeletion.⑵.Therearetwotypesoferrorcontrolsa.presyntheticerrorcontrol.b.proofreadingcontrol⑶.mismatchrepair.SectionTwoFeaturesofDNAPolymerases1.ThesubstratesofDNApolymerasesare2.TheactivecenterofDNApolscatalyzesDNAsynthesis.⑴.TheactivecentercandifferentiatedNTPfromNTP.⑵.DNApolscanchoosetherightnucleotideforbase-pairingwiththetemplatenucleo-tide.3.Thesemi-closedright-handedstructureoftheDNApol.iscomposedofthreedomains.⑴.thumbdomain,fingersdomainandpalmdomain⑵.twoactivecenters:polymeraseactivecenterand3’-5’exonucleaseactivecenter.Theyarelocatedinthepalmdomain.⑶.Thepalmdomainhasthreefunctions:a.polymeraseactivityb.tocheckthenewlyformedbasepairc.proofreadingcontroltoremovethe‘mis-paired‘nucleotide.⑷.Thefingersdomainbindsthetemplatestrandandinteractswiththenucleotidethatentersthepolymeraseactivecenter.⑸.Thethumbdomainkeepstheprimer-templatejunctioninpositionintheactivecenterandm