分子生物学7-DNA-damage-repair-and-recombinant

DNAdamage,repairandrecombinationDNA损伤、修复和重组Chapter6DamageandMutation（损伤和突变）DNAdamageissimplyachemicalalterationtoDNA.DNA损伤仅是DNA分子的化学改变AmutationisaheritablepermanentchangeinthebasesequenceofDNA.DNA碱基序列水平上的永久性可遗传的改变。UnrepairedDNAdamagecanleadtomutation.未修复的DNA损伤可导致突变Thenatureofmutations(突变的本质)Pointmutation点突变（Substitution碱基置换）Mutationsthatalterasinglenucleotide.Transition(转换):AGTCTransversion(颠换):ATorCTAorGGTorCCAorGFrameshiftmutation移码突变Insertion插入Deletion缺失Theexamplesofmutations突变的例子-T-C-G-G-C-T-G-T-A-C-G--A-G-C-C-G-A-C-A-T-G-C-transition转换-T-C-G-A-G-C-T-G-T-A-C-G--A-G-C-T-C-G-A-C-A-T-G-C-Insertion插入A-T-C-G-C-T-G-T-A-C-G--A-G-C-G-A-C-A-T-G-C-Deletion缺失TWildtype野生型基因-T-C-G-A-C-T-G-T-A-C-G--A-G-C-T-G-A-C-A-T-G-C--T-C-G-T-C-T-G-T-A-C-G--A-G-C-A-G-A-C-A-T-G-C-transversion颠换碱基对的置换(substitution)移码突变(frameshiftmutation)Base-pairsubstitutionSilentmutationsNonsensemutationsMissensemutations沉默突变无义突变错义突变Phenotypiceffects表型效应Basepairdeletionorinsertionnonsensemutations无义突变missensemutations错义突变Phenotypiceffects表型效应missensemutations错义突变Anexampleofmutation突变的一个例子Threeimportantsourcesofmutations三种重要的突变源•InaccuracyinDNAreplicationandmeioticrecombination•ThechemicaldamagestoDNA•Theclassofinsertionsgeneratedbytransposons（转座子）Spontaneousmutation自发突变AlterationsinDNAsequencecausedbyspontaneouserrorsinDNAreplicationormeioticrecombination指DNA复制或减数分裂的重组中自发产生的错误引起的生物DNA序列的改变Theoccurrencerateofspontaneousmutationsisverylow,dependingonthedifferentgenes.自发突变发生率很低，而且不同基因的发生率也不同Virusandbacterialgenes病毒和细菌基因1/1010～1/108Humangenes人类基因1/106～1/105Mousegenes小鼠基因1/105～1/104Replicationfidelity复制忠实性1.DNApolymerase（DNA聚合酶）2.Repairsystem修复系统Functions功能Pol.IPol.IIPol.III引发链合成---5’-3’polymeriation+++3’-5’exonucleaseactivity+++5’-3’exonucleaseactivity+--Properbasepairing，proofreading，mismatchrepair，RNAprimingTheperpetuationofthegeneticmaterialsfromgenerationtogenerationdependsonmaintainingratesofmutationatlowlevel.遗传物质代代相传依赖于突变概率维持在低水平上。However,ifthegeneticmaterialswereperpetuatedwithperfectfidelity,thegeneticvariationneededtodriveevolutionwouldbelacking,andnewspecieswouldnothavearisen.然而，传给子代的遗传物质如果绝对精确的话，就失去了驱动进化的基因变异，新的物种也不会形成。Threeimportantsourcesofmutations三种重要的突变源•InaccuracyinDNAreplicationandmeioticrecombination•ThechemicaldamagestoDNASpontaneoushydrolysisanddeaminationMutagenesisPhysicalmutagenesisChemicalmutagenesis•TheclassofinsertionsgeneratedbytransposonsDNAdamagespontaneouslyfromhydrolysisanddeaminationDepurinationsites脱嘌吟位点A,G,CorTBreakoftheglycosylicbondcausesthelostofbases,leavingthesitesfreeofbases,towhichanynucleotidescouldbeinsertedduringDNAreplicationMutagenesis诱变-1Physicalmutagenesis物理诱变Physicalmutagens物理诱变剂IonizingradiationbreakofDNAdoublestrands,whichisdifficulttoberestored.X-andγ-rayNonionizingradiationmolecularvibrationorthepromotionofelectronstohigherenergylevels,leadingtotheformationofnewchemicalbonds.UVMutagenesiscausedbyradiationisnonspecificandoccursatrandom.Mutagenesis诱变-2Chemicalmutagenesis化学诱变Chemicalmutagens化学诱变剂Baseanalogs:derivativesofthenormalbaseswithalteredbasepairingproperties.DirectmutagenesisNitrousacidsdeaminationofcytosine,adenineAlkylatingandarylatingagentslesionIndirectmutagenesisIntercalatorsinsertionanddeletionmutationBaseanalogs碱基类似物AG5-溴尿嘧啶(5-BU)5-溴去氧尿核苷(5-BudR)2-氨基嘌吟(2-AP)5-BUcanpairtobothAandG,causingerrorinbasepairs,whichwillresultintransition.Alkylatingagents烷化剂Guanineisethylatedinto6-ethylguanine，whichcanbase-pairwiththymineEMS甲基磺酸乙酯，硫酸二乙酯（DES）等，带有一个或多个活泼的烷基，能碱基形成烷基化碱基，改变氢键的结合能力，从而改变碱基的配对。AcridinedyesinserttoDNAB-DNAbeforeintercalationB-DNAafterintercalationIntercalatingagents2-氨基吖啶、吖啶橙、ICR-170等能嵌入DNA双链中的碱基之间，引起单一核酸的缺失或插入，造成突变。UVandhighenergyradiation紫外线和高能量放射物Threeimportantsourcesofmutations三种重要的突变源•InaccuracyinDNAreplicationandmeioticrecombination•ThechemicaldamagestoDNA•Theclassofinsertionsgeneratedbytransposons（转座子）DNArepairDNA修复Photoreactivation光复活Alkyltransferaserepair烷基转移酶修复Excisionrepair切除修复Mismatchrepair错配修复SOSrepairSOS修复Homologousrecombination同源重组Howtofind?Howtorepair?Photoreactivation光复活作用Cleavageofthecyclobutanering(环丁烷环)ofpyrimidinedimersbyDNAphotolysesrestorestheoriginalDNAstructure.Error-freedirectreversal无错直接逆转Alkyltransferaserepair烷基转移酶修复Thealkylgroupisremovedfromalkylatedbasebyalkyltransferaseandtransferedtotheenzymeitself.Error-freedirectreversal无错直接逆转Baseexcisionrepair碱基切除修复APsiteBaseexcisionrepairNucleotideexcisionrepair核苷酸切除修复Excinuclease（UvrABC）NickNickDNApolymeraseIDNAligaseExcisionendonuclease，anuclease(enzyme)whichexcisesafragmentofnucleotidesduringDNArepair12-13ntMismatchrepair错配修复GATCCTAGATGATCCTAGACGATCCTAGATGATCCTAGATMismatchrepaira,unmethylatedGATCis5’ofmutationb,unmethylatedGATCis3’ofmutationSOSrepair紧急修复DNArecombinationHomologousrecombination1同源重组112RecBCDendonucleasenicks2strandsrecBCDΧsite”（chisite）5'-GCTGGTGG-3‘①同源分子联会DNAsequenceareidenticalornearlyidenticalforaregionofatleast100bporso.②DNA断裂Homologousrecombination2同源重组23Strandexchangeiscalled“strandinvasion”,accomplishedbyrecAprotein,strand-exchangeprotein链侵入Homologousrecombination3同源重组3Singl