The Hereditary Material
Discovery of the Hereditary Material
• Mendel’s work was rediscovered ~1903
• The chromosome theory of inheritance was
proposed in ~1903
– chromosomes behave much like Mendel’s
“factors”
• DNA was discovered in the mid-1800’s
– “simple repetitive sequences of A, C, T, G”
– “too simple to store complex information”
Discovery of the Hereditary Material
• chromosomes are ~50% DNA & 50% protein
– proteins are enormously complex
• for a 20n a.a. protein: 20, 400, 8000,
160,000…
–20100 = 1.3130
– proteins were “obviously” the hereditary
material
Zeroing in on DNA
• a “transforming principle” occurs in some
living organisms - Griffith, 1928
– pneumococcus (Streptococcus pneumoniae)
• virulent strain - capsule; smooth colonies
• avirulent strain - no capsule; rough
colonies
Figure 11.1
Zeroing in on DNA
• a “transforming principle” occurs in some
living organisms - Griffith, 1928
– Avery et al. (1944) analyzed it chemically
• extracted biomolecules
• degraded each class
• tested for transformation
–without DNA => no transformation
–with DNA => transformation
structure of a
bacteriophage
Figure 11.2
Zeroing in on DNA
• Hershey & Chase (1952) hosted a DNA/
protein showdown with bacteriophage (made
exclusively of DNA and protein)
– labeled phage DNA with 32P
or
– labeled phage protein with 35S
then
– infected bacteria with each
32P
remained with
(in) the bacteria;
35S
was “knocked
off” the bacteria
and,
progeny viruses
contained 32P,
but not 35S
Figure 11.3
X-ray crystallography
Figure 11.4
The Nature of DNA
• X-ray crystallography - Franklin & Wilkins,
1950
– DNA is a helical molecule
• Chargraff’s chemical analysis of DNA, 1950
– purines = pyrimidines
• %A + %G = %T + %C
Chargraff’s Rules
Figure 11.5
The Nature of DNA
• X-ray crystallography - Franklin & Wilkins,
1950
– DNA is a helical molecule
• Chargraff’s chemical analysis of DNA, 1950
– purines = pyrimidines
• %A + %G = %T + %C
• model building - Watson & Crick, 1953
– interpretation of accumulated data &
application of organic chemistry
d.s. DNA structure
Figure 11.6
DNA base pairs
DNA backbone
antiparallel backbones
Figure 11.7
The Nature of DNA
• model building - Watson & Crick, 1953
– double helix
– complementary base pairs
– sugar-phosphate backbones
– DNA helices are antiparallel
The Nature of DNA
• DNA structure should explain biological
processes
– DNA replication - exact copies of each
chromosome
• three possible modes
–dispersive
–conservative
–semi-conservative
semiconservative
or
conservative
or
dispersive
Figure 11.8
CsCl gradient centrifugation - experimental
grown in 15N
grown in 14N
Figure 11.9
DNA-dependent DNA synthesis
Figure 11.10
The Nature of DNA
• DNA structure should explain biological
processes
– DNA replication - semi-conservative
• each strand serves as a template for a new
strand
• each new strand grows from 5’=>3’ by the
addition of base-paired dNTPs
Figure 11.13
Replication Model
Figures 11.11, 11.12
Replication of DNA
• DNA replication requirements
– template DNA
– dNTPs
– DNA polymerase
– ORIgin of Replication
• local denaturation
• initiation of synthesis
– primer
DNA
polymerase
requires
a primer
(a free
3’-OH)
Figure 11.14
machinery at the replication fork
Figure 11.15
DNA replication
• DNA polymerase
– DNA polymerase III catalyzes leading
strand and lagging strand synthesis
– DNA polymerase I removes RNA primers
• DNA ligase seals gaps in the sugar-phosphate
backbone of the lagging strand
Okazaki
fragments
leave
gaps
in the
lagging
strand
Figure
11.16
DNA polymerase I removes
RNAprimers;
DNA ligase seals
gaps in lagging strand
Figure 11.17
telomerase adds to the 3’
end of the lagging strand
template
Figure 11.18
DNA Proofreading & Repair
• replication occurs at 1000-2000 bp per second
• replication occurs with high fidelity
– <1 error in 108 to 1012 nucleotides
– replication machinery is not that reliable
Proofreading and Repair
proofreading by DNA polymerase
during replication
mismatch repair
during or soon after replication
excision repair
before or after S phase
Figure 11.19
during or soon after replication
DNA Proofreading & Repair
• proofreading & repair increase fidelity
– proofreading - by DNA polymerase
– mismatch repair - corrects errors using the
template strand
– excision repair
• corrects chemical damage,
insertions/deletions, etc.
–cut a section of the offending strand
–remove the flawed region
–repair with DNA pol I & DNA ligase
Polymerase Chain Reaction (PCR)
• a lab technique to replicate DNA quickly
– start with a double stranded template
– add primers complementary to the 3’ ends of
each template
– add dNTPs
– add DNA polymerase
– get - two copies of the DNA between the
primers
Polymerase Chain Reaction (PCR)
• Repeat
– start with original template and new strands
– primers are already there
– dNTPs are already there
– DNA polymerase is already there
– get - four copies of DNA between primers
– …8 copies, 16 copies, 32 copies…
– >1 million/20 cycles; >1 billion/30 cycles
DNA amplification by PCR
Figure 11.20
Sanger’s sequencing secret: ddNTPs
automated Sanger sequencing
Figure 11.21
DNA sequencing
• Sanger dideoxynucleotide sequencing strategy
– start with many template copies
– add dNTPs
– add labeled ddATP
– add DNA polymerase
– get - lots of new strands, each ending with a
ddATP at a different T on the template
strand
– repeat with each ddNTP
– high resolution gel
DNA sequencing
• automate Sanger dideoxy-sequencing
– start with different colored ddNTPs in the
same tube
– analyze by high resolution gel
electrophoresis read by a robot