分子生物学Chapter 5DNA Damage and Repair.docx

分子生物学Chapter 5DNA Damage and Repair.docx

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分子生物学Chapter5DNADamageandRepair

Chapter5DNADamageandRepair

Introduction

TheDNAsequencecanbechangedastheresultofcopyingerrorsintroducedbyDNApolymerasesduringreplicationandbyenvironmentalagentssuchasmutagenicchemicalsandcertaintypesofradiation.IfDNAsequencechanges,whatevertheircause,areleftuncorrected,bothgrowingandnongrowingsomaticcellsmightaccumulatesomanymutationsthattheycouldnolongerfunction.Inaddition,theDNAingermcellsmightincurtoomanymutationsforviableoffspringtobeformed.ThusthecorrectionofDNAsequenceerrorsinalltypesofcellsisimportantforsurvival.

TherelevanceofDNAdamageandrepairtothegenerationofcancer（carcinogenesis）becameevidentwhenitwasrecognizedthatallagentsthatcausecancer（carcinogens）alsocauseachangeintheDNAsequenceandthusaremutagens.AlltheeffectsofcarcinogenicchemicalsontumorproductioncanbeaccountedforbytheDNAdamagethattheycauseandbytheerrorsintroducedintoDNAduringthecells'effortstorepairthisdamage.Likewise,ultraviolet（UV）radiationandionizingradiation（x-raysandatomicparticles）notonlymodifyDNA,butalsocancausecancerinanimalsandcantransformnormalcellsincultureintorapidlyproliferating,cancer-typecells.Theabilityofionizingradiationtocausehumancancer,especiallyleukemia,wasdramaticallyshownbytheincreasedratesofleukemiaamongsurvivorsoftheatomicbombsdroppedinWorldWarII,andmorerecentlybytheincreaseinmelanoma（skincancer）inindividualsexposedtotoomuchsunlight.

1.Mutagenesis

vMutation

vReplicationfidelity

vMutagens:

chemical&physical

vMutagenesis:

direct&indirect

Mutation

vPermanent,heritablealterationsinthebasesequenceofDNA

Reasons

vSpontaneouserrorsinDNAreplicationormeioticrecombination

vAconsequenceofthedamagingeffectsofphysicalorchemicalmutagensonDNA

Pointmutation

（asinglebasechange）

Transition:

PurineorpyrimidineisreplacedbytheotherAGTC

Transversion:

apurineisreplacedbyapyrimidineorviceverse

vATorCTAorG

vGTorCCAorG

Insertionsordeletions:

TheadditionorlossofoneormorebasesinaDNAregion

Frameshiftmutations:

TheORFofaproteinencodedgeneischangedsothattheC-terminalsideofthemutationiscompletelychanged

Amutationinvolvingachangeinasinglebasepair,oftencalledapointmutation,adeletionorinsertionofafewbasepairsgenerallyaffectsthefunctionofasinglegene.

Changesinasinglebasepairmayproduceoneofthreetypesofmutation（Figure5-1a）:

•Missensemutation,whichresultsinaproteininwhichoneaminoacidissubstitutedforanother

•Nonsensemutation,inwhichastopcodonreplacesanaminoacidcodon,leadingtoprematureterminationoftranslation

•Frameshiftmutation,whichcausesachangeinthereadingframe,leadingtointroductionofunrelatedaminoacidsintotheprotein,generallyfollowedbyastopcodon

Smalldeletionshaveeffectssimilartothoseofframeshiftmutations,althoughonethirdofthesewillbein-frameandresultinremovalofasmallnumberofcontiguousaminoacids.

Thesecondmajortypeofmutationinvolveslarge-scalechangesinchromosomestructureandcanaffectthefunctioningofnumerousgenes,resultinginmajorphenotypicconsequences.

Suchchromosomalmutations（orabnormalities）caninvolvedeletionorinsertionofseveralcontiguousgenes,inversionofgenesonachromosome,ortheexchangeoflargesegmentsofDNAbetweennonhomologouschromosomes（Figure5-1b）.

Figure5-1.Differenttypesofmutations.（a）Pointmutations,whichinvolvealterationinasinglebasepair,andsmalldeletionsgenerallydirectlyaffectthefunctionofonlyonegene.Awild-typepeptidesequenceandthemRNAandDNAencodingitareshownatthetop.Alterednucleotidesandaminoacidresiduesarehighlightedingreen.Missensemutationsleadtoachangeinasingleaminoacidintheencodedprotein.Inanonsensemutation,anucleotidebasechangeleadstotheformationofastopcodon（purple）.Thisresultsinprematureterminationoftranslation,therebygeneratingatruncatedprotein.Frameshiftmutationsinvolvetheadditionordeletionofanynumberofnucleotidesthatisnotamultipleofthree,causingachangeinthereadingframe.Consequently,completelyunrelatedaminoacidresiduesareincorporatedintotheproteinpriortoencounteringastopcodon.（b）ChromosomalabnormalitiesinvolvealterationsinlargesegmentsofDNA.Presumablytheseabnormalitiesariseowingtoerrorsinthemechanismsforrepairingdouble-strandbreaksinDNA.Chromosomes（IorII）areshownassinglethicklineswiththeregionsinvolvedinaparticularabnormalityhighlightedingreenorpurple.Inversionsoccurwhenabreakisrejoinedtothecorrectchromosomebutinanincorrectorientation;deletions,whenasegmentofDNAislost;translocations,whenbreaksarerejoinedtothewrongchromosomes;andinsertions,whenasegmentfromonechromosomeisinsertedintoanotherchromosome.

1.2Replicationfidelity

vImportantforpreservethegeneticinformationfromonegenerationtothenext.

vMutationrelevant

SpontaneouserrorsinDNAreplicationisveryrare,oneerrorper1010baseinE.coli.

Molecularmechanismsforthereplicationfidelity

vDNApolymerase:

Watson-Crickbasepairing

v3’5’proofreadingexonuclease.

vRNApriming:

proofreadingthe5’endofthelaggingstrand

vMismatchrepair

2.DNADamage

2.1MutationsOccurSpontaneously

MutationsarisespontaneouslyatlowfrequencyowingtothechemicalinstabilityofpurineandpyrimidinebasesandtoerrorsduringDNAreplication.DNAdoesundergomajorchangesasaresultofthermalfluctuations.Wenowknow,forexample,thatabout5000purinebases（adenineandguanine）arelostperdayfromtheDNAofeachhumancellbecauseofthethermaldisruptionoftheirN-glycosyllinkagestodeoxyribose（depurination）.Similarly,spontaneousdeaminationofcytosinetouracilinDNAisestimatedtooccuratarateof100basespergenomeperday（Figure5-2）.DNAbasesarealsosubjecttochangebyreactivemetabolites（includingreactiveformsofoxygen）thatcanaltertheirbase-pairingabilitiesMostofthemwouldbeexpectedtoleadeithertodeletionofoneormorebasepairsinthedaughterDNAchainafterDNAreplicationortoabase-pairsubstitution（eachC→Udeamination,forexample,wouldeventuallychangeaC-GbasepairtoaT-Abasepair,sinceUcloselyresemblesTandformsacomplementarybasepairwithA）.（Figure5-3）

AnothercauseofspontaneousmutationsiscopyingerrorsduringDNAreplication.Althoughreplicationgenerallyiscarriedoutwithhighfidelity,errorsoccasionallyoccur.Figure5-4illustrateshowonetypeofcopyingerrorcanproduceamutation.Intheexampleshown,themutantDNAcontainsnineadditionalbasepairs.

Figure5-2.Deaminationanddepurination.ThesehydrolyticreactionsarethetwomostfrequentspontaneouschemicalreactionsknowntocreateseriousDNAdamageincells.Onlyasingleexampleisshownforeachtypeofreaction.（SeealsoFigure6-39.）

Figure5-3.Formationofaspontaneouspointmutationbydeaminationofcytosine（C）toformuracil（U）.IftheresultingU·GbasepairisnotrestoredtothenormalC·Gbasepairbyrepairmechanisms,itwillbefixedintheDNAduringreplication.Afteroneroundofreplication,onedaughterDNAmoleculewillhavethemutantU·Abasepairandtheotherwillhavethewild-typeC·Gbasepair.Theuracilisremovedandreplacedbythymine,generatingamutantDNAinwhichaT·ApairreplacesaC·Gpair.

Figure5-4.OnemechanismbywhicherrorsinDNAreplicationproducespontaneousmutations.Thereplicationofonlyonestrandisshown;theotherstrandisreplicatednormally,asshownatthetop.AreplicationerrormayariseinregionsofDNAcontainingtandemlyrepeatedsequences（inthiscase,GTC）whenaportionofthenewlysynthesizedstrand（lightblue）loopsoutintoasingle-strandedform.Thisslippagedisplacesthenewlysynthesizedstrandbackalongthetemplatestrand（darkblue）,withits3′endstillpairedwiththetemplate.Asaresult,theDNA-synthesizingenzymescopyaregionofthetemplatestrandasecondtime,leadingtoanincreaseinlengthofninenucleotides（yellow）inthisexample.AsubsequentroundofDNAreplicationresultsintheproductionofonenormalduplexDNAmoleculeandonemutantduplexcontainingtheadditionalnucleotides.

SomeHumanDiseasesAreCausedbySpontaneousMutations:

Manycommonhumandiseases,oftendevastatingintheireffects,areduetomutationsinsinglegenes.Geneticdiseasesarisebyspontaneousmutationsingermcells（eggandsperm）,whicharetransmittedtofuturegenerations.Forexample,sickle-cellanemia,whichaffects1in500individualsofAfricandescent,iscausedbyasinglemissensemutationatcodon6oftheβ-globingene;asaresultofthismutation,theglutamicacidatposition6inthenormalproteinischangedtoavalineinthemutantprotein.Thisalterationhasaprofoundeffectonhemoglobin,theoxygen-carrierproteinoferythrocytes,whichconsistsoftwoα-globinandtwoβ-globinsubunits.Thedeoxygenatedformofthemutantproteinisinsolubleinerythrocytesandformscrystallinearrays.Theerythrocytesofaffectedindividualsbecomerigidandtheirtransitthroughcapillariesisblocked,causingseverepainandtissuedamage.Becausetheerythrocytesofheterozygousindividualsareresistanttotheparasitecausingmalaria,whichisendemicinAfrica,themutantallelehasbeenmaintained.ItisnotthatindividualsofAfricandescentaremorelikelyt