Download DNA Structure and Analysis

History
Fred Griffith – 1928
Streptococcus pneumonia – bacterial
pneumonia
Two strains
Smooth (S) – capsule: virulent pathogen
Rough (R) – non-virulent
S
mouse died
R
mouse lived
Heat-killed S + R
mouse died
blood
(smooth live bacteria)
Transforming principle
Griffith’s Transformation
Experiment
History
 Friedrich Miescher – 1869
 Isolated DNA
 T.H. Morgan
 Genes located on chromosomes
 DNA and proteins became candidates for genetic
material

Proteins took the lead: heterogeneity and specific function
History
 Max Delbrück and Salavador Luria – 1940
 Bacteriophage (bacteria eaters)
 E. coli phages T1-T7
 Easy to work with, reproduce every 25 minute
 Oswald Avery 1944
 Purified various chemicals from pathogenic bacteria;
tried to transform live non-virulent bacteria
 DNA is the transforming principle
 Greeted with skepticism
History
 Erwin Chargaff – 1950
 DNA composition varies from one species to another
 % ratio of nucleotides (A=T and G = C)
 Hershey and Chase – 1952
 Blender experiment


S-32 - proteins
P-35 - DNA
Blender Experiment
Blender Experiment
Blender Experiment
Watson and Crick
 Nature: 1953
 X-ray crystallography - Franklin/Wilkens
 Helical shape, width of molecule, composed of two strands
(double helix)
 Chargaff Base Pairing Rule
DNA Structure
 Nucleotide: building block (Monomer)
 5-carbon sugar: deoxyribose
 Phosphate group
 Nitrogenous base

Adenine, Guanine, Thymine, Cytosine
Antiparallel
chains run in opposite directions
DNA
Molecule
Double Helix
Antiparallel
chains run in opposite directions
5′ end
phosphate attached to 5′
deoxyribose carbon
3′ end
hydroxyl attached to 3′
deoxyribose carbon
Base Pairing
Hydrogen bonding
• Occurs between base pairs;
binds the two DNA strands
• Adenine (A) with thymine (T)
form two hydrogen bonds
• Guanine (G) with cytosine (C)
form three hydrogen bonds
DNA Replication
Meselson-Stahl Experiment
Semi-conservative replication
• E. coli grown in medium
containing heavy nitrogen
(15N): incorporated 15N
into DNA
Transferred from 15N to 14N
medium
after two generations, DNA
density supported
semiconservative replication
Semiconservative replication
Parental DNA
First generation
Second generation
Fig. 12-7a, p. 268
Semi-conservative Replication
Each daughter double helix
consists of
1 original strand from
parent molecule
1 new complementary
strand
Adenine
Deoxyribose
Guanine
Deoxyribose
Thymine
Deoxyribose
Cytosine
Deoxyribose
Fig. 12-6b, p. 267
DNA Replication
Origins of Replication
-
-
specific nucleotide sequence where replication
begins
Attachment site for proteins that initiate
replication – forms a replication bubble
Replication is bi-directional
Eukaryotic cells have multiple origins of replication
-Strands separate
- Helicases: open the double helix
- Single-strand binding proteins (SSB): keep
strands separated and stabilize DNA
Bi-directional Replication
DNA Replication
Synthesis of new strand:
- DNA polymerase: catalyzes elongation of DNA
strand
- adds nucleotides to the free 3/ end;
DNA strand grows in a 5/ to 3/ direction
- 53 Rule
- energy provided by nucleoside
triphosphate
DNA Replication
Always proceeds in 5′ → 3′ direction
Leading strand
synthesized continuously
Lagging strand
synthesized discontinuously
forms short Okazaki fragments (100-200
nucleotides)
DNA primase synthesizes RNA primers
DNA ligase links Okazaki fragments
DNA Replication
Priming
DNA polymerase can only add to a pre-existing
chain – which is the RNA primer
Short RNA strand (5-14 nucleotides)
Removed later