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1、DNA replication(复制复制)1.OverviewA parental duplex of DNA is replicated to form two daughter duplexes,each of which consists of one parental strand and one(newly synthesized)daughter strand.This behavior is called semiconservative(半保留半保留)replication.1.1 conceptsThe replicon(复制子复制子)is a unit of the gen
2、ome in which DNA is replicated.Each replicon contains an origin for initiation of replication.The origin(起始点起始点)is a sequence of DNA at which replication is initiated.The terminus(终点终点)is a segment of DNA at which replication ends.A replication fork(Growing point)is the point at which strands of par
3、ental duplex DNA are separated so that replication can proceed.A replication eye is a region in which DNA has been replicated within a longer,unreplicated region.Semidiscontinuous replication is mode in which one new strand is synthesized continuously while the other is synthesized discontinuously.O
4、kazaki fragments are the short stretches produced during discontinuous replication;they are later joined into a covalently intact strand.Leading strand:ContinuousLagging strand:Discontinuous Okazaki fragmentsSingle-replicon vs Multi-replicon Unidirection vs BidirectionLinear DNA vs circular DNASingl
5、e strand vs double strands1.2 Models of DNA replicationTheta()mode replicationWhen a replicon is circular,the presence of an eye forms the-structure Displacement loop replication/D loop replicationRolling circle replication1.3 Priming is required to start DNA synthesis A primer is a short sequence(o
6、ften of RNA)that is paired with one strand of DNA and provides a free 3-OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.A sequence of RNA synthesized on the template(cellular DNA)A preformed RNA pairs with the template(retrovirus)A primer terminus generated within du
7、plex DNA.(rolling circle replication)priming nucleotide provided by protein bound to DNA(certain viruses)2.Replication in Bacteria2.1 Initiation of replicationvRecognition of originvThe two strands of DNA initially separate,which is a melting reaction over a short region.vAn unwinding point begins t
8、o move along the DNA;this marks the generation of the replication fork.vThe first nucleotides of the new chain must be synthesized into the primer.The origin is a sequence of DNA at which replication is initiated.Initiating a replication cycle.Controlling the frequency of initiation events.A general
9、 feature origins is AT-rich,assumedly related to DNA melting.13bp repeats:9bp repeats:Initiation of replication at oriC starts with formation of a complex that requires six proteins:DnaA,DnaB,DnaC,HU,Gyrase,and SSB.DnaA:recognition of origin of replicationDnaB:DNA helicase that unwinds parental dupl
10、exDnaC:loads helicase(DnaB)onto DNASSB:maintains DNA single-stranded stateGyrase:type II topoisomerase,relieves positive supercoil ahead of replication forkHU:a histone-like protein,prevents nonspecific initiation at sites other than oriC.The primase(primerase)is a type of RNA polymerase that synthe
11、sizes short segments of RNA that will be used as primers for DNA replication.The primosome is a protein complex capable of synthesizing RNA primers on single stranded DNA during DNA replication,main components of which are helicase and primerase.The primosome is utilized once on the leading strand o
12、f DNA and repeatedly,initiating each Okazaki fragment,on the lagging DNA strand.2.2 Elongation of replicationThe replisome is the multiprotein structure that assembles at the replicating fork to undertake synthesis of DNA.It contains DNA polymerase and other enzymes.E.coli PolymeraseIIIIIIPolymerase
13、 gene*Pol APol BPol CSubunits(number of different types)1410MW103,00088,000(polB)830,000DNA polymerase+3-5 Exonuclease(proofreading)+5-3 Exonuclease+-Rate(nt/sec)16-2040150-400Processivity(nts added before polymerase dissociates)3-2001500 500,000Functionremoval of primer&repairrepairreplication2.2.1
14、 common structure and proofreading mechanism of DNA polymerases -Pol I&Pol IIIDNA polymerase IC-terminal larger fragmentN-terminal small fragment(Klenow fragment)Protease digestDNA polymerase35 exonuclease53 exonucleaseNick translation(切口平移切口平移)describes the ability of E.coli DNA polymerase I to use
15、 a nick as a starting point from which one strand of a duplex DNA can be degraded and replaced by resynthesis of new material;is used to introduce radioactively labeled nucleotides into DNA in vitro.DNA polymerase IC-terminal larger fragmentN-terminal small fragment(Klenow fragment)Protease digestDN
16、A polymerase35 exonuclease53 exonucleasecatalytic active sitepositioning the template correctly at the active sitebinds the DNA as it exits the enzymeExonuclease sitePolymerase active site35 exonuclease active siteFidelity of replication10-5-10-610-210-1-10-2Accurate selection of nucleotides10-9Immediate proofreadingPostreplicative mismatch repairSpontaneous mutations occur in the absence of any added reagent to increase the mutation rate,as the result of errors in replication(or other events involved in the reproduction of DNA)or by environmental damage.