现代分子生物学之DNA复制教学提纲.ppt

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1、现代分子生物学之DNA复制AnnealedprimerTheChemistryofDNASynthesistemplateΔG=-3.5kcal/molΔG=-7kcal/molTheMechanismofDNAPolymerasePalm:foradditionofdNTPsandforremovalofmispaireddNTP.(1)Bindstotwometalions----alterthechemicalenvironment-----catalysis.(2)H-bondswithminorgrooveofDNA----Monitorstheaccuracy

2、ofthenewbase-pairFinger:BindstothenewdNTP;Bendsthetemplate;StabilizethepyrophosphateThumb:Holdstheprimer-templatejunctioninactivesite,reducesrateofdissociationtemplatestrandprimerstranddNTPNucleotideaddition:catalyzedattheactivesiteofDNApolymeraseH-bondcontactsbetweenminorgroove(newBP)and

3、palmTwometalionsareheldinplacebyinteractionswithtwohighlyconservedaspartateresidues.Nucleotideaddition:catalyzedattheactivesiteofDNApolymeraseH-bondcontactsbetweenminorgroove(newBP)andpalmTwometalionsareheldinplacebyinteractionswithtwohighlyconservedAsp.MetalionAinteractswiththe3'-OH→redu

4、cetheOandHassociation→leavesanucleophilic3'O-.MetalionBinteractswithTPsofthenewdNTPtoneutralizetheirnegativecharge.Aftercatalysis,thePPiisstabilizedbyinteractingwithmetalionBNucleotideaddition:catalyzedattheactivesiteofDNApolymeraseH-bondcontactsbetweenminorgroove(newBP)andpalmFingerdomai

5、nbindstotheincomingdNTPandbendsthetemplateFingerdomainbindstotheincomingdNTPandbendsthetemplateFingerdomainbindstotheincomingdNTPandbendsthetemplateMovetoenclosedNTPFingerdomainbindstotheincomingdNTPandbendsthetemplateDNApolymerase“grips”thetemplateandtheincomingdNTPwhenacorrectbasepairis

6、mademetalionsThecriticaltyr,lys,argThebaseanddeoxyriboseofthedNTPTheprimerTemplatestrandPhosphatesStericconstraints空间位阻preventsDNApolfromusingrNTPprecursors.Howtoavoidincorporationofwrongnucleotideswrongbasepairlowersrateofcatalysis(kineticselectivity)准--稳--快--接；错--晃--慢--掉Eachbaseshasitsp

7、referredtautomericformTheoccasionalflickingofthebasesinto“wrong”tautomericform:10-5mistakeExonucleasesproofreadnewlysynthesizedDNATheaveragenumberofnucleotidesaddedeachtimetheenzymebindsaprimer-templatejunctionTheprocessivityofDNAPolymerasevariesfromafewto>50,000nuc