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LECTURE 12: DNA: STRUCTURE & REPLICATION I
CH7 key concepts
genetic material: DNA
DNA structure
DNA replication
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
DNA = GENETIC MATERIAL
Griffith (1928), Streptococcus pneumoniae
transforming principle in bacteria is DNA
DNA = GENETIC MATERIAL
Avery et al. (1944), Streptococcus pneumoniae
more proof ... transforming principle is DNA
DNA = GENETIC MATERIAL
Hershey & Chase (1952), T2 bacteriophage
DNA (not protein) is genetic material
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?
DNA STRUCTURE
chemical structure of the 4 nucleotides in DNA
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
DNA STRUCTURE
bonding is specific: pyrimidines bond with purines
implication: mechanism for accurate replication
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
DNA STRUCTURE
clues for Watson & Crick (1953) ... X-ray data
& ratios ( A + G ) = ( T + C ) ... Chargaff
DNA STRUCTURE
DNA double helix (space filling model)
several forms... A, B & Z DNA
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
DNA REPLICATION
3 possible models of DNA replication:
DNA REPLICATION
Meselson & Stahl (1958), tested the models
DNA REPLICATION
what would have happened if...
DNA REPLICATION
plant root cells incubated with [3H] thymidine
colchicine to
inhibit spindles
(chromosomes
do not separate)
autoradiography
DNA REPLICATION
explanation of [3H] thymidine incubation / colchicine
treatment / autoradiography at DNA level
DNA REPLICATION
“harlequin chromosomes”, incubated with
bromodeoxyuridine (BUdR)
DNA REPLICATION
harlequin chromosomes of Chinese hamster ovary
cells, incubated with bromodeoxyuridine (BUdR)
chromatid
exchange
DNA REPLICATION
semiconservative
replication
linear gene
arrangement
... chromatid =
single DNA
molecule +
protein matrix
chromatid
exchange
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)
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
DNA REPLICATION
replication fork ?
bacteria incubated with [3H] thymidine...
daughter molecules  1 “hot” + 1 “cold” strand
autoradiograph...
DNA REPLICATION
replication... theta () structures
replication fork
DNA REPLICATION
some molecules use rolling-circle replication
MECHANISM OF DNA REPLICATION
Kornberg (late ‘50s)
enzyme that catalyzes DNA replication ...
DNA polymerase I
several others
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
MECHANISM OF DNA REPLICATION
prokaryotes have a single origin of replication
bidirectional replication with 2 forks  single terminus
~ 20 minutes (*... book says 40)
MECHANISM OF DNA REPLICATION
E. coli origin of replication
tandem repeat consensus sequence
protein binding sites
MECHANISM OF DNA
REPLICATION
replication to completion...
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 ...
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