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CH7 key concepts

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Title: CH7 key concepts


1
LECTURE 12 DNA STRUCTURE REPLICATION I
  • CH7 key concepts
  • genetic material DNA
  • DNA structure
  • DNA replication

2
CHAPTER 7 KEY CONCEPTS
  • transforming principle in bacteria is DNA
  • experiments with labeled T2 phage establish that
    DNA is the hereditary material
  • Watson Crick DNA model anti-parallel double
    helix nucleotides linked by phosphodiester
    bonds helices are held together by hydrogen
    bonds between pairs of nitrogenous bases
  • DNA structure ensures fidelity of replication
    base pairing specified by hydrogen bonding
  • DNA replication is semiconservative daughter
    duplex has 1 parental 1 new strand
  • DNA replication depends on many enzymes

3
DNA GENETIC MATERIAL
  • Griffith (1928), Streptococcus pneumoniae
  • transforming principle in bacteria is DNA

4
DNA GENETIC MATERIAL
  • Avery et al. (1944), Streptococcus pneumoniae
  • more proof ... transforming principle is DNA

5
DNA GENETIC MATERIAL
  • Hershey Chase (1952), T2 bacteriophage
  • DNA (not protein) is genetic material

6
DNA GENETIC MATERIAL
  • why was the scientific community reluctant to
    accept DNA (rather than protein) as hereditary
    material?...
  • DNA is a simple molecule...
  • how is complexity of life
    encoded by such simplicity?
  • how is information accurately transmitted
    from one generation to the
    next?

7
DNA STRUCTURE
  • chemical structure of the 4 nucleotides in DNA

8
DNA STRUCTURE
  • hydrogen bonded nucleotides on opposite helices
  • DNA helices are antiparallel
  • carbons on sugar define ends... 5' and 3'
  • pyrimidines bond with purines
  • T ? A
  • C ? G

9
DNA STRUCTURE
  • bonding is specific pyrimidines bond with
    purines
  • implication mechanism for accurate replication

10
DNA STRUCTURE
  • helical structure of DNA
  • major minor groves
  • 10Å radius 20Å diameter
  • 3.4Å between nucleotide base pairs
  • 34Å / 360 turn
  • 10 nucleotide base pairs / 360 turn

11
DNA STRUCTURE
  • clues for Watson Crick (1953) ... X-ray data
  • ratios ( A G ) ( T C ) ... Chargaff

12
DNA STRUCTURE
  • DNA double helix (space filling model)
  • several forms... A, B Z DNA

13
DNA REPLICATION
  • model of replication proposed by Watson Crick
    (1953)
  • parental strand template
  • semiconservative model (new double helix has 1
    template 1 new daughter strand)
  • 3 possible models
  • conservative
  • semiconservative
  • dispersive

14
DNA REPLICATION
  • 3 possible models of DNA replication

15
DNA REPLICATION
  • Meselson Stahl (1958), tested the models

16
DNA REPLICATION
  • what would have happened if...

17
DNA REPLICATION
  • plant root cells incubated with 3H thymidine
  • colchicine to
    inhibit spindles
    (chromosomes

    do not separate)
  • autoradiography

18
DNA REPLICATION
  • explanation of 3H thymidine incubation /
    colchicine treatment / autoradiography at DNA
    level

19
DNA REPLICATION
  • harlequin chromosomes, incubated with
    bromodeoxyuridine (BUdR)

20
DNA REPLICATION
  • harlequin chromosomes of Chinese hamster ovary
    cells, incubated with bromodeoxyuridine (BUdR)

chromatid exchange
21
DNA REPLICATION
  • semiconservative replication
  • linear gene arrangement
  • ... chromatid single DNA molecule protein
    matrix

chromatid exchange
22
DNA REPLICATION
  • model of replication proposed by Watson Crick
    (1953)
  • parental strand template
  • semiconservative model (new double helix has 1
    template 1 new daughter strand)

23
DNA REPLICATION
  • model of replication proposed by Watson Crick
    (1953)
  • parental strand template
  • semiconservative model (new double helix has 1
    template 1 new daughter strand)
  • replication fork

24
DNA REPLICATION
  • replication fork ?
  • bacteria incubated with 3H thymidine...
  • daughter molecules ? 1 hot 1 cold strand
  • autoradiograph...

25
DNA REPLICATION
  • replication... theta (?) structures
  • replication fork

26
DNA REPLICATION
  • some molecules use rolling-circle replication

27
MECHANISM OF DNA REPLICATION
  • Kornberg (late 50s)
  • enzyme that catalyzes DNA replication ...
    DNA polymerase I
  • several others

28
MECHANISM OF DNA REPLICATION
  • DNA replication requirements...
  • 1. H bonds between bases must be broken
  • 2. chain separation/unwinding
  • 3. available pools of 4 dNTPs A T, C ? G
  • 4. enzymes
  • E. coli...
  • 4.7 107 bp (47,000,000)
  • molecule has 4.7 106 turns (10 bp/turn)
  • 1500 bp/s, 20 min to divide with 1 origin

29
MECHANISM OF DNA REPLICATION
  • prokaryotes have a single origin of replication
  • bidirectional replication with 2 forks ? single
    terminus
  • 20 minutes (... book says 40)

30
MECHANISM OF DNA REPLICATION
  • E. coli origin of replication
  • tandem repeat consensus sequence
  • protein binding sites

31
MECHANISM OF DNA REPLICATION
  • replication to completion...

32
MECHANISM OF DNA REPLICATION
  • eukaryotes have multiple origins of replication
  • bidirectional replication with 2 forks
  • 1.4 h (yeast) to 24 h (cultured animal cells)...
    or more
  • issues...
  • linear vs circular
  • coordination of multiple chromosomes
  • pulse/chase and autoradiography ...

33
MECHANISM OF DNA REPLICATION
  • eukaryotes have multiple origins of replication
  • bidirectional replication with 2 forks
  • 1.4 h (yeast) to 24 h (cultured animal cells)...
    or more
  • issues...
  • linear vs circular
  • coordination of multiple chromosomes
  • pulse/chase and autoradiography ... Drosophila
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