[生物学]chapter 5 dna replication.ppt

Chapter 5 DNA Replication,Contents,An Overview Bacterial DNA replication Eukaryotic DNA replication Telomere replication Rolling circle replication,5.1 Basic mechanism and enzymology,1. semi-conservative mechanism 2. Replicons 3. Bidirectional replication 4. semi-discontinuous replication 5. RNA priming 6. Enzymology of DNA replication,1. Semi-conservative Replication,(1) Definition: DNA replication in which the two strands of parental duplex separate completely, each acting as a template for synthesis of a complementary strand . One parental strand is therefore conserved in each progeny duplex.,(2) semi-conservative replication hypotheses 1953 Watson & Crick: DNA double helix model Semi-conservative Replication (3) Three hypotheses for DNA replication,,,,Parental strands,Semi-conservative,Conservative,Dispersive,Daughter strands,(4) Meselson-Stahl’s experiment（1958）,,,,,,,,,,14N-14N,14N-15N,15N-15N,15NH4Cl,14NH4Cl,,,,,20 min,40 min,,,Summary for the experiment: DNA replicates in a semiconservative manner. When the parental strands separate, each serves as the template for making a new, complementary strand.,Semi-conservative,2. Replicons,(1) Replicon is a unit of the genome in which DNA is replicated. Each replicon contains an origin for initiation of replication. Origin is the DNA sequence where replication is initiated. Terminus is the DNA sequence where replication ends.,(2) Prokaryotic replicon,Prokaryotic genome: a single circular DNA , a single replicon,,,,,,,origin,terminus,,,,,(3) Eukaryotic replicons,The long, linear DNA molecules of eukaryotic chromosomes consist of mutiple replicons, each with its own origin.,,,,,,3. Bidirectional replication,Most eukaryotic and prokaryotic DNAs replicate bidirectionally.,,,,,origin,terminus,,,,,,,Bidirectional replication describes a system in which an origin generates two replication forks that proceed away from the origin in opposite directions. Unidirectional replication refers to the movement of a single replication fork from a given origin.,A replication eye is a region in which DNA has been replicated within a longer, unreplicated region. A replication fork is the point at which strands of parental duplex DNA are separated so that replication can proceed.,,,,,,,,Continuous, semidiscontinuous, and discontinuous models of DNA replication,4. Semi-discontinuous Replication,1968年 Okazaki Semi-discontinuous model of DNA replication (1) Definition: Semi-discontinuous replication is a mode in which one new strand is synthesized continuously while the other is synthesized discontinuously.,,(2) Discovery of Okazaki fragments---Evidence for semi-discontinuous replication,[3H] thymidine pulse-chase labeling experiment Add [3H] thymidine in the medium for a few second→ break the cell → analyzed on alkaline sucrose gradient→ found a large fraction of nascent DNA (1000-2000 nt) = Okazaki fragments Grow the cell in regular medium then → analyze → the small fragments join into high molecular weight DNA = Ligation of the Okazaki fragments,Okazaki fragments are the short stretches of 1000-2000 bases produced during discontinuous replication; they are later joined into a covalently intact strand. The leading strand of DNA is synthesized continuously in the 5’–3’ direction. The lagging strand of DNA must grow overall in the 3’-5’ direction and is synthesized discontinuously in the form of short fragments (5’-3’ ) that are later connected covalently.,Summary DNA replication in E. coli (and in other organisms) is semi-discontinuous. One strand is replicated continuously in the direction of the movement of the replicating fork; the other is replicated discontinuously as 1-2 kb Okazaki fragments in the opposite direction. This allows both strands to be replicated in the 5’-3’ direction.,5. RNA priming,(1) Primer: 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.,(2) Function provides a free 3’-OH end to initiate DNA synthesis Crucial for high fidelity of replication,(3) Priming activity Priming activity is required to provide 3’–OH ends to start off the DNA chains on both the leading and lagging strands. The leading strand requires only one such initiation event, which occurs at the origin. But there must be a series of initiation events on the lagging strand. Each Okazaki fragment starts with a primer sequence of RNA that provides the 3’–OH end for extension by DNA polymerase.,(4) The primase is a type of RNA polymerase that synthesizes short segments of RNA that will be used as primers for DNA replication. Primosome: a mobile complex which includes the helicase and primase and synthesizes RNA primers every 1000-2000 nt on the lagging strand. primase: Priming leading strand synthesis primosome: Priming lagging strand synthesis (5) Primer removal Primers are removed, subsequent gaps filling with DNA and ligation.,6. DNA polymerase in E. coli,。