dna replication and repair - ms. kim...•dna polymerases that carry out replication also play...
TRANSCRIPT
DNAReplicationandRepairLesson2
Semiconservative
replication
• AmechanismofDNA
replicationinwhicheach
ofthetwostrandsof
parentDNAis
incorporatedintoanew
strandofDNA
DNAReplicationOverview
Three steps:
1. SeparationoftheparentDNAstrands
2. Buildingofcomplementary(daughter)strands
3. Proofreadingandrepairingerrorsinthenewstrands
Step1– StrandSeparation
• TheReplicationorigin isaspecificsequenceofDNA=thestartingpointforreplication
• TheY-shapedstructurethatformsasthetwostrandsseparateisknownasthereplicationfork
Twochallenges:
1. Tension iscreated
aheadofthefork
2. Tendencytore-
join,oranneal
(complementary)
1. Topoisomerases relievestensioninDNAstrandsby
cuttingoneortwostrandsnearthefork,allowing
strandstountangle,andthenre-jointhecutstrands
2. Single-strandbindingproteins(SSBs) prevent
annealingbyattachingtoDNAstrandstostabilize
themandkeepthemseparated
Single-strandbindingproteins(SSBs)
•HelicaseenzymebindstotheseoriginsarebeginstounwindthetwostrandsofDNAfromtheorigin(canseparateinbothdirections)bybreakingH-bondsb/wcomplementarybasepairs
• Astheforksproceedinoppositedirections,thespacebetweenthemiscalledareplicationbubble,soonfilledwiththenewlyreplicatedDNA
Step2– BuildingComplementaryStrands
NewnucleotidesarejoinedbyagroupofenzymescalledDNApolymerases(prokaryoteexamplewillbediscussed)
• DNApolymerases ”read”thetemplatestrandinits3’à5’direction
• Theycanonlyaddnucleotidestothe3’endofanexistingDNAstrand
• Thus,thenewstrandisalwaysassembledinthe5’to3’direction
• RNAprimase enzymesbeginthereplicationprocessby
buildingasmallcomplementaryRNAsegmentcalledRNA
primers(10-60ribonucleotideslong)
• DNApolymeraseIII beginstoaddDNAnucleotidestotheprimer
• SinceDNApolymeraseIIIonlybuilds5’à3’,thetwonewstrandsbegintobeassembledinoppositedirections
• DNApolymeraseIIIisabletocontinuecontinuously
• NoneedfortheRNAprimasetoaddadditionalprimers
• Thisiscalledtheleadingstrand
• Ontheoppositestrand,DNApolymeraseIIIismovingawayfromthereplicationforkà laggingstrand
• RNAprimaseattachesanotherprimerallowingDNApolymeraseIIItobeginfromanewpoint
• ThepatterncreatedonthesecondstrandisaseriesofRNAprimersandshortDNAfragmentscalledOkazakifragments
• Aseachfragmentextendsinthe5’à3’direction,iteventuallyrunsintotheRNAprimerattachedtotheOkazakifragmentahead
•DNApolymeraseI removestheRNAnucleotidesandreplacesthemwithDNAnucleotides
•DNAligasecatalyzestheformationsofphosphodiesterbondstosealthestrand
Step3– DealingwithErrors(repairing)
• DNApolymerasesthatcarryoutreplicationalsoplayanotherimportantrole
• AstheyassemblenewDNAstrands,theyproof-readandcorrecterrors(base-pairmismatches)
• Proof-reading:Whilecreatingthecomplementarystrand,ifamismatchoccurs,DNApolymeraseIIImaybackup,repair,andcontinue
• Repairing:DNArepairmechanismsofproteinsandenzymes(suchasDNAPolymeraseIandII)maylocatedistortionsinthestrandsbetweenreplicationeventsandremoveapieceofthestrand,DNApolymeraseswillfillthegap,andDNAligasewillsealthestrand
Proof-reading
• DNApolymeraseIIIcontinuesaddingnucleotidesintheforwarddirection• Iftheenzymeaddsamismatchednucleotide,theenzymeactsasaexonuclease(cleavenucleotides)toremovethemismatchednucleotide• TheenzymeresumesactivityasDNApolymerase
Repairing
• DNApolymeraseII repairsdamagetoDNAthatoccursbetweenreplicationevents• Repaircomplexesremoveseveraltomanybases,leavingagapintheDNA• GapisfilledinbyaDNApolymerase,usingthetemplateasaguide• NickissealedbyDNAligasetocompleterepair
Homework
Investigation6.4.1
•WorkingroupstocreatearoleplayforDNAreplication
(role-playandquestionsdueFridayNov17)