分子生物學(xué) DNA.ppt

1、DNA replication (復(fù)制),1. Overview,A 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) replication.,1.1 concepts,The replicon (復(fù)制子) is a unit of th

2、e genome in which DNA is replicated. Each replicon contains an origin for initiation of replication.,The origin (起始點(diǎn)) is a sequence of DNA at which replication is initiated. The terminus (終點(diǎn)) is a segment of DNA at which replication ends.,A replication fork (Growing point) is the point at which stra

3、nds of parental 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 disc

4、ontinuously. Okazaki fragments are the short stretches produced during discontinuous replication; they are later joined into a covalently intact strand.,Leading strand: Continuous Lagging strand: Discontinuous Okazaki fragments,Single-replicon vs Multi-replicon,Unidirection vs Bidirection,Linear DNA

5、 vs circular DNA,Single strand vs double strands,1.2 Models of DNA replication,Theta () mode replication,When a replicon is circular, the presence of an eye forms the -structure,Displacement loop replication /D loop replication,Rolling circle replication,1.3 Priming is required to start DNA synthesi

6、s,A primer is a short sequence (often 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

7、),A primer terminus generated within duplex DNA. (rolling circle replication),priming nucleotide provided by protein bound to DNA (certain viruses),2. Replication in Bacteria,2.1 Initiation of replication,Recognition of origin The two strands of DNA initially separate, which is a melting reaction ov

8、er a short region. An unwinding point begins to move along the DNA; this marks the generation of the replication fork. The 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. Contro

9、lling the frequency of initiation events.,A general 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 or

10、igin of replication DnaB: DNA helicase that unwinds parental duplex DnaC: loads helicase (DnaB) onto DNA SSB: maintains DNA single-stranded state,Gyrase: type II topoisomerase, relieves positive supercoil ahead of replication fork HU: a histone-like protein, prevents nonspecific initiation at sites

11、other than oriC.,The primase (primerase) is a type of RNA polymerase that synthesizes 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

12、 are helicase and primerase. The primosome is utilized once on the leading strand of DNA and repeatedly, initiating each Okazaki fragment, on the lagging DNA strand.,2.2 Elongation of replication,The replisome is the multiprotein structure that assembles at the replicating fork to undertake synthesi

13、s of DNA. It contains DNA polymerase and other enzymes.,2.2.1 common structure and proofreading mechanism of DNA polymerases -Pol I is used to introduce radioactively labeled nucleotides into DNA in vitro.,DNA polymerase I,C-terminal larger fragment,N-terminal small fragment,(Klenow fragment),Protease digest,DNA polymerase 35 exonuclease,53 exonuclease,Polymerase active site,35 exonuclease active site,Fidelity of replication,10-5-10-6,10-2,10-1-10-2,Accurate selection of nucleotides,10-9,Immediate proofreading,Postreplicative mismatch repai