生物化学 经典课件 15DNA复制和重组 考研必备 学生.ppt

*（一）DNA的半保留复制DNA半保留复制的实验证据（二）DNA复制的起点和方式D环复制1956年Kornberg发现的DNA聚合酶I（100kg大肠杆菌可以分离0.5g纯化的酶）由一条肽链组成，有5′→3′聚合酶活力，5′→3′外切酶活力，和3′→5′外切酶活力。用蛋白酶水解得到的Klenow片段有3′→5′外切酶活力和5′→3′聚合酶活力。图示为Klenow片段与DNA的结合，模板链(12nt)为蓝色，引物链(14nt)为红色。DNA聚合酶I的5′3′外切酶可以从单链切口的5′末端切除核苷酸，5′3′聚合酶可以用脱氧核苷酸填补缺口。DNA聚合酶Ⅲ的β亚基二聚体与DNA结合的空间关系(顶部观)。DNA连接酶的作用机制（四）DNA的半不连续复制（五）DNA复制的拓扑性质复制的起始大肠杆菌DNA复制的步骤大肠杆菌DNA复制时冈崎片段的合成步骤复制的延伸复制的终止（七）真核生物DNA的复制
DNAreplicationineukaryoticcellsuseessentiallythesameprinciplesbutbeingmorecomplexinthedetails.
ThebestunderstoodeukaryoticreplicationsystemisthatofSV40andyeast.
FormationoftheRNAprimersandtheinitialincorporationofdeoxynucleotidesarebelievedtobecatalyzedbyDNApolymeraseα(whichhasnoproofreadingactivity).
LaterchainextensionisbelievedtobecatalyzedbyDNApolymeraseδ,whichhasproofreadingactivityandisclampedontotheDNAtemplatesviathePCNAtrimerrings.Specificsequences(~150bp)thatcanfunctionasreplicationoriginshaveonlybeenidentifiedinyeast(iscalledautonomouslyreplicatingsequences,orARS)andSV40virus.
TherateofDNAsynthesisineukaryoticcellsisaboutonetenthofthatoftheE.colicells.
Replicationintheeukaryoticgenomesbeginatmanyreplicationorigins.
9purifiedproteinsareneededtoreplicateSV40virusDNAinvitro.
Tantigen：amultifunctionalsite-specificDNAbindingproteinencodedbySV40DNA,bindstotheorigin(asDnaA)andmeltstheduplexDNA(asDnaB);RPA：encodedbythehostmammaliancellsandbindstothemeltedsingle-strandedDNA(asSSB).
Polα/primase：synthesizestheRNAprimersandastretchofDNAsequences,hasnoproofreadingactivity.
Polδ–replacesPolα/primasetofurtherextendtheRNA-DNAstrand,hasproofreadingactivity.
PCNA(proliferatingcellnuclearantigen)：atrimericring-shapedproteinthatclampsPolδontotheDNAtemplate.
RFC(replicationfactorC)：aclamploaderforPCNA.
Topoisomerases：Relievesthetorsionalstraininducedbythegrowingreplicationfork.
Ligases：joinstheOkazakifragments(whichismuchshorterthanthoseinE.colicells),aswellastheleadingstrand.几种真核细胞的DNA聚合酶的性质如表中所示，体外或者是体内的实验均证明，DNA聚合酶α和δ可以被抑制剂2＇3＇-双脱氧胸苷和蚜肠霉素抑制，这些结果可以说明它们是DNA的复制酶。E.coliλSV40/人酵母功能
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