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)