Chapter 20 DNA Replication I: Basic Mechanism and Enzymology CopyrightThe McGraw-Hill Companies,Inc.Pemmission required for reproduction or display Ken Eward/SS/Photo Researchers.Inc
Chapter 20 DNA Replication I: Basic Mechanism and Enzymology
20.1 General Features of DNA Replication 1 DNA replication is semiconservative (a)Semiconservative (b)Conservative (c)Dispersive
1 DNA replication is semiconservative 20.1 General Features of DNA Replication
Meselson and Stahl (1958)Demonstration of Semiconservative Replication Matthew Messelson Franklin Stahl
Matthew Messelson Franklin Stahl Meselson and Stahl (1958) Demonstration of Semiconservative Replication
NI5 NI4 Ni4 wash transfer wash transfer G0 G1 G2 E.coli first grown on a heavy isotope of Nitrogen (N15)in Generation 0(GO) then bacteria washed and transferred to the lighter isotope N14 for both Generation 1(G1)and Generation 2(G2). Old DNA incorporated heavy isotope,while newly synthesized DNA must incorporate the light isotope
✓E. coli first grown on a heavy isotope of Nitrogen (N15) in Generation 0 (G0) ✓then bacteria washed and transferred to the lighter isotope N14 for both Generation 1 (G1) and Generation 2 (G2). ✓ Old DNA incorporated heavy isotope, while newly synthesized DNA must incorporate the light isotope. wash & transfer wash & transfer N15 N14 N14 G0 G1 G2
Copyright The McGraw-Hill Companies,Inc.Permission required for reproduction or display. Figure 20.2 14N 15N (a) (b) Separation of DNA by cesium chloride density gradient centrifugation CsCI密度梯度离心分离DNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Separation of DNA by cesium chloride density gradient centrifugation CsC1密度梯度离心分离DNA
First generation (a) onservative: + HH HH ↓ Expected density gradient results: HH HH First Second generation generation () Semiconservative 一+一+++ ↓↓ Expected density gradient results: T First Second generation generation (@) ispersive: -+川一川+川+川+川 HH mixed Expected density gradient results: D二
Figure 20.4 (a) (b) Generations 0.3 0.7 Results of CsCl gradient 1.0 ultracentrifugation 1.1 experiment to demonstrate 1.5 semiconservative M 1.9 DNA replication 2.5 3.0 4.1 0 and 1.9 mixed 0 and 4.1 mixed Melon and pcol
Results of CsCl gradient ultracentrifugation experiment to demonstrate semiconservative DNA replication
15N-DNA Heat- denatured 0 15N-DNA 0o000e Heat-denatured! hybrid DNA Heat- Heat- denatured denatured 15N-DNA 14N-DNA Density→
Photo- DNA in E.coli graphs Densito- CsCl DNA of DNA metric cultures gradient Start composition bands scans 15N-containing Heavy medium <DNA Continue growing first generation in 14N medium Replication cycle 2 Heavy,light (hybrid 15N/14N) DNA Continue Proof DNA growing Replication Replication Semi- cycle 3 Light Heavy, (14N/n4N light conservative DNA (15NW14N)） DNA Continue growing Replication cycle 4 14N/14N 14N/14N 14N/14N 15N/14N N15-red strand H4N DNA 4N DNA 2 A N14-black strand
Proof DNA Replication Semiconservative N15 - red strand N14 - black strand
There are various“problems”that must be overcome for DNA polymerase to copy DNA polymerases ->catalyze nucleotide addition at the 3' hydroxyl end of a growing chain,so strands can grow only in the 5'to 3'direction -Okazaki fragment polymerases -only elongate a preexisting DNA or RNA strand (the primer)and are unable to initiate chains -RNA primers polymerases -unable to melt duplex DNA in order to separate the two strands that are to be copied helicase and SSB
There are various “problems” that must be overcome for DNA polymerase to copy DNA • polymerases →catalyze nucleotide addition at the 3- hydroxyl end of a growing chain, so strands can grow only in the 5 to 3 direction - Okazaki fragment • polymerases → only elongate a preexisting DNA or RNA strand (the primer) and are unable to initiate chains - RNA primers • polymerases → unable to melt duplex DNA in order to separate the two strands that are to be copied - helicase and SSB