04-2 Biosynthesis of DNA--Replication

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1、§4TheBiosynthesisofDNA---Replication(2)DNAsynthesisrequiresdeoxynucleosidetriphosphatesandaprimer：templatejunctionDNAissynthesizedbyextendingthe3’endoftheprimerHydrolysisofpyrophosphate(PPi)isthedrivingforceforDNAsynthesisReviewSubstraterequiredforDNAsynthesis§4-5Enzymology

2、ofDNAReplicationReplisomeisthemultiproteinstructurethatassemblesatthereplicationforktoundertakesynthesisofDNA.ContainsDNApolymeraseandotherenzymes.1.DoubleStrandsSeparationItisthefirststepofDNAreplicationthatdoublestrandsseparateintosinglestrand.Threekindsofproteinareimport

3、antinthisprocess.1)Helicase:AhelicaseisanenzymethatseparatesthestrandsofDNA,usuallyusingthehydrolysisofATPtoprovidethenecessaryenergy.E.colihelicase:DnaB,PriAandRepprotein.Helicasealsojoinsintheprocessofrepairing.2)Single-strandbindingprotein(SSB):Asingle-strandbindingprote

4、in(SSB)bindstothesingle-strandedDNA,preventingitfromreformingtheduplexstate.TheSSBbindsasamonomer,buttypicallyinacooperativemannerinwhichthebindingofadditionalmonomerstotheexistingcomplexisenhanced.SSBalsojoinsintheprocessofrepairing.3)Topoisomerase(gyrase):负责产生负超螺旋并使两个子代环状

5、DNA分子分开。Single-strandedbindingproteins(SSBs)stabilizesingle-strandedDNA2.DNApolymeraseAnenzymethatcansynthesizeanewDNAstrandonatemplatestrandiscalledaDNApolymerase.ProkaryticDNApolymeraseE.coliDNApolymeraseⅠ，proofreadingremovesRNAprimersⅡ，repairingⅢ，replicationIV，SOSV，SOS1）

6、E.coliDNApolymeraseE.coliDNApolymeraseI：monomer，109kDa，928AA。用枯草杆菌酶或胰蛋白酶消化，可获得大小两个片段：Largefragment--KlenowI:C端324~928AA,5’-3’polymeraseand3’-5’exonuclease.Smalldomain:5’-3’exonucleaseProofreadingNicktranslationProofreading:Proofreadingreferstoanymechanismforcorrectingerrors

7、inproteinornucleicacidsynthesisthatinvolvesscrutinyofindividualunitsaftertheyhavebeenaddedtothechain.3’-5’exonuclease5’-3’polymeraseExonucleasesproofreadnewlysynthesizedDNATheoccasionalflickingofthebasesinto“wrong”tautomericformresultsinincorrectbasepairandmis-incorporatio

8、nofdNTP.(10-5mistake)ThemismatcheddNMPisremovedbyproofreadingexonuclease,apartofth