Download 13. DNA Replication

Biology 212 General Genetics
Lecture 13 "DNA Replication"
Spring 2007
Reading: Chap. 6 pp. 210-224
Lecture Outline:
1. DNA structure review
2. Semiconservative replication model
3. Replication forks
4. Process of replication
a. Key enzymes and requirements
b. Leading and lagging strands
Lecture:
1. Review of DNA structure
DNA double helix model:
 DNA made of nucleotide building blocks linked into polymer chains
 Bases are on inside, sugars and phosphates form a backbone on outside
 Two strands exist in an antiparallel arrangement
5'----------- 3'
3'----------- 5'
 hydrogen bonds between the complementary bases A pairs with T; G pairs with C
2. Semiconservative model of DNA replication
 in simplest form, the concept that each strand of DNA serves as the template for
synthesis of the daughter strand (Fig. 6.8)
experimental evidence for the model
Meselson and Stahl 1958

Experimental system
o E. coli bacterium
 One large double stranded circular chromosome
 Duplicates itself in about 30 min.
o Minimal growth requirements
 Glucose (carbon source)
glucose minimal
 Ammonium salts (nitrogen source)
medium
 Various salts
 Water
o Used heavy (15N) or normal (14N) isotopes in different minimal media.
o Separate 15N-tagged DNA from 14N DNA by equilibrium density gradient
centrifugation
1
14
15
N-DNA (less dense)
N-DNA (more dense)
Experiment
1. grow bacteria for many generations in 15N-containing medium (heavy)
2. transfer bacteria to 14N-containing medium (light)
3. isolate DNA from bacteria periodically and determine relative density on equilibrium
density gradient.
4. interpretation of results based on model of semi-conservative replication
Figs. 6.9 and 6.10
3. Replication forks
a. Replication begins at origins of replication
E. coli bacterial DNA
 1 unique origin
 bidirectional replication from origin
 replication bubble (theta structure) intermediate
higher organisms
 multiple replication origins
b. Replication forks


sites where double stranded DNA strands have separated
new DNA is laid down
4. Process of DNA replication
 multistep process
 catalyzed by many enzymes and proteins
 requires deoxynucleotide triphosphates
a. Key steps and key enzymes: E. coli replication
Key steps
1. unwind DNA at origin
keep DNA unwound
relieve stress on DNA from
unwinding
2. lay down short RNA primer
Key enzymes and proteins
helicase
single-stranded DNA binding proteins (SSB)
DNA gyrase
RNA polymerase (primase)
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3. extend primer with dNTPs in
5'  3' direction
proofread to remove mispaired
bases
4. remove RNA primer and
replace with DNA
5. seals nicks in DNA backbone
so strand is continuous
DNA polymerase III
DNA polymerase I
DNA ligase
b. DNA replication is discontinuous
 DNA is copied in pieces then assembled
 Due to unique nature of DNA polymerase enzymes--only work in 5' 3' direction
 Discovered by Okazaki
 One strand is synthesized continuously=leading strand
 One strand is synthesized discontinuously=lagging strand
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