Chapter 16~ The Molecular Basis of

Inheritance
 Scientific History
• The march to understanding that DNA is
the genetic material
– T.H. Morgan (1908)
– Frederick Griffith (1928)
– Avery, McCarty & MacLeod (1944)
– Erwin Chargaff (1947)
– Hershey & Chase (1952)
– Watson & Crick (1953)
– Meselson & Stahl (1958)
 The “Transforming Principle”1928

• Frederick Griffith
– Streptococcus pneumonia bacteria
• was working to find cure for pneumonia
– harmless live bacteria (“rough”) mixed
with heat-killed pathogenic bacteria
(“smooth”) causes fatal disease in
mice
– a substance passed from dead
bacteria to live bacteria to change
their phenotype
• “Transforming Principle”
 The “Transforming Principle”
mix heat-killed
pathogenic &
live pathogenic live non-pathogenic heat-killed non-pathogenic
strain of bacteria strain of bacteria pathogenic bacteria bacteria
A. B. C. D.

mice die mice live mice live mice die

Transformation = change in phenotype

something in heat-killed bacteria could still transmit
disease-causing properties
 DNA is the “Transforming
1944
Principle”
• Avery, McCarty & MacLeod
– purified both DNA & proteins separately from
Streptococcus pneumonia bacteria
• which will transform non-pathogenic bacteria?
– injected protein into bacteria
• no effect
– injected DNA into bacteria
• transformed harmless bacteria into
virulent bacteria

mice die

What’s the
conclusion?
Avery Experiment
 1944 | ??!!
Avery, McCarty & MacLeod
• Conclusion
– first experimental evidence that DNA was the genetic
material

Oswald Avery Maclyn McCarty Colin MacLeod

 1952 | 1969
Confirmation of DNA Hershey

• Hershey & Chase

– classic “blender” experiment
– worked with bacteriophage
• viruses that infect bacteria
– grew phage viruses in 2 media,
Why use radioactively labeled with either
Sulfur
vs.
• 35
S in their proteins
Phosphorus?• 32
P in their DNA
– infected bacteria with
labeled phages
 Protein coat labeled DNA labeled with 32P
with 35S
Hershey T2 bacteriophages
are labeled with
& radioactive isotopes
S vs. P

Chase bacteriophages infect

bacterial cells

bacterial cells are agitated

Which to remove viral protein coats
radioactive
marker is found
inside the cell?

Which molecule
carries viral 35
S radioactivity 32
P radioactivity found
genetic info? found in the medium in the bacterial cells
 Blender experiment
• Radioactive phage & bacteria in blender
– 35S phage
• radioactive proteins stayed in supernatant
• therefore viral protein did NOT enter bacteria
– 32P phage
• radioactive DNA stayed in pellet
• therefore viral DNA did enter bacteria
– Confirmed DNA is “transforming factor”

Taaa-Daaa!
 1952 | 1969
Hershey & Chase Hershey

Martha Chase Alfred Hershey

 Chargaff 1947

• DNA composition: “Chargaff’s rules”

– varies from species to species
– all 4 bases not in equal quantity
– bases present in characteristic ratio
• humans: Rules
A= 30.9%
A=T
C=G
T = 29.4%
G = 19.9%
C = 19.8%
That’s interesting!
What do you notice?
 Structure of DNA 1953 | 1962

• Watson & Crick

– developed double helix model of DNA
• other leading scientists working on question:
– Rosalind Franklin
– Maurice Wilkins
– Linus Pauling

Franklin Wilkins Pauling

 1953 article in Nature
Watson and Crick

Watson Crick
Rosalind Franklin (1920-1958)
 Double helix structure of DNA

“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
material.” Watson & Crick
 Directionality of DNA
• You need to PO4 nucleotide
number the
carbons!
– it matters! N base

5 CH2
This will be O
IMPORTANT!!
4 ribose 1

3 2
OH
The DNA backbone 5
PO4
• Putting the DNA
base
backbone together 5 CH2

– refer to the 3 and 5 O

4 1
ends of the DNA C
3 2
• the last trailing carbon O
–
O P O
Sounds trivial, but…
O base
this will be 5 CH
2
IMPORTANT!! O
4 1

3 2
OH
3
Anti-parallel strands
• Nucleotides in DNA
backbone are bonded from
phosphate to sugar between 5 3
3 & 5 carbons
– DNA molecule has “direction”
– complementary strand runs in
opposite direction

3 5
 Bonding in DNA
hydrogen
bonds
5 3

covalent
phosphodiester
bonds

3
5

….strong or weak bonds?

How do the bonds fit the mechanism for copying DNA?
 Base pairing in DNA
• Purines
– adenine (A)
– guanine (G)
• Pyrimidines
– thymine (T)
– cytosine (C)
• Pairing
–A:T
• 2 bonds
–C:G
• 3 bonds
 But how is DNA copied?
• Replication of DNA
– base pairing suggests that
it will allow each side to
serve as a template for a
new strand

“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
material.” — Watson & Crick
 Copying DNA
• Replication of DNA
– base pairing allows
each strand to serve as
a template for a new
strand
– new strand is 1/2 parent
template &
1/2 new DNA
• semi-conservative
copy process
Semiconservative replication,
• when a double helix replicates each of the daughter molecules will
have one old strand and one newly made strand.
• Experiments in the late 1950s by Matthew Meselson and Franklin
Stahl supported the semiconservative model, proposed by Watson
and Crick, over the other two models. (Conservative & dispersive)
 Let’s meet

DNA Replication
the team…

• Large team of enzymes coordinates replication

 Replication: 1st step
• Unwind DNA
– helicase enzyme
• unwinds part of DNA helix
• stabilized by single-stranded binding proteins
helicase

single-stranded binding proteins replication fork

 Replication: 2nd step
 Build daughter DNA
strand
 add new
complementary bases
 DNA polymerase III

DNA
Polymerase III
 5 3
Replication
energy
DNA
• Adding bases Polymerase III

– can only add energy

nucleotides to DNA
Polymerase III
3 end of a growing
DNA strand energy
DNA
• need a “starter” Polymerase III
nucleotide to
bond to energy
DNA
– strand only grows Polymerase III

53
3 5
 Okazaki

Leading & Lagging strands

Limits of DNA polymerase III
 can only build onto 3 end of an
existing DNA strand 5


rag ments
ki f
Okaza 5
3 5 5 3
3
5 Lagging strand
3
ligase
growing 3
replication fork
5
Leading strand

Lagging strand

3 5

3

 Okazaki fragments DNA polymerase III

 joined by ligase Leading strand
 “spot welder” enzyme  continuous synthesis
 Replication fork / Replication
3 bubble 5

5 3

DNA polymerase III

leading strand
5
3
3 5
5 5
5 3 3
lagging strand

3 5
5
3 lagging strand leading strand growing
5
3 replication fork 5
5 growing
replication fork 5
leading strand 3
lagging strand
3
5
5 5
 Starting DNA synthesis: RNA
primers
Limits of DNA polymerase III
 can only build onto 3 end of an
existing DNA strand 5

3 5 3
5
3
3 5

growing 3 primase
replication fork DNA polymerase III
5

RNA 5

RNA primer 3
 built by primase
 serves as starter sequence for DNA
polymerase III
 Starting DNA synthesis: RNA
primers
Limits of DNA polymerase III
 can only build onto 3 end of an
existing DNA strand 5

3 5 3
5
3
3 5

growing 3 primase
replication fork DNA polymerase III
5

RNA 5

RNA primer 3
 built by primase
 serves as starter sequence for DNA
polymerase III
 Replacing RNA primers with DNA
DNA polymerase I
 removes sections of RNA primer and
replaces with DNA nucleotides DNA polymerase I
5

3

3
5 ligase
growing 3
replication fork
5

RNA 5

3

But DNA polymerase I still

can only build onto 3 end of
an existing DNA strand
 Houston, we
have a problem!

Chromosome erosion
All DNA polymerases can
only add to 3 end of an DNA polymerase I
existing DNA strand 5

3

3
5
growing 3
replication fork DNA polymerase III
5

RNA 5

Loss of bases at 5 ends 3

in every replication
 chromosomes get shorter with each replication
 limit to number of cell divisions?
 Telomeres
Repeating, non-coding sequences at the end
of chromosomes = protective cap
 limit to ~50 cell divisions 5

3

3
5
growing 3 telomerase
replication fork
5

5

Telomerase TTAAGGGTTAAGGGTTAAGGG 3
 enzyme extends telomeres
 can add DNA bases at 5 end
 different level of activity in different cells
 high in stem cells & cancers -- Why?
 Replication fork
DNA
polymerase III lagging strand
DNA
polymerase I
3’
Okazaki primase
fragments 5’
5’ ligase
3’ 5’ SSB

3’ helicase

DNA
polymerase III
5’ leading strand
3’
direction of replication
SSB = single-stranded binding proteins
 DNA polymerases
• DNA polymerase III
Roger Kornberg
– 1000 bases/second! 2006
– main DNA builder
• DNA polymerase I
– 20 bases/second
– editing, repair & primer removal
DNA polymerase III Arthur Kornberg
enzyme 1959
 Editing & proofreading DNA
• 1000 bases/second =
lots of typos!
• DNA polymerase I
– proofreads & corrects typos
– repairs mismatched bases
– removes abnormal bases
• repairs damage
throughout life
– reduces error rate from
1 in 10,000 to
1 in 100 million bases
 Fast & accurate!
• It takes E. coli <1 hour to copy
5 million base pairs in its single
chromosome
– divide to form 2 identical daughter cells
• Human cell copies its 6 billion bases &
divide into daughter cells in only few hours
– remarkably accurate
– only ~1 error per 100 million bases
– ~30 errors per cell cycle
What does it really look like?

4
Any Questions??

2007-2008
DNA Packing
DNA
double
helix
(2-nm
diameter

Histones

“Beads on
a string”

Nucleosome
(10-nm diameter)

Tight helical fiber

(30-nm diameter) Supercoil
(200-nm diameter)
700
nm

Metaphase chromosome
Nucleosomes 8 histone
molecules
• “Beads on a string”
– 1st level of DNA packing
– histone proteins
• 8 protein molecules
• positively charged amino acids
• bind tightly to negatively charged
DNA
DNA packing as gene
control
• Degree of packing of DNA regulates
transcription
– tightly wrapped around histones
• no transcription  heterochromatin
• genes turned off darker DNA (H) = tightly packed
 euchromatin
lighter DNA (E) = loosely packed

H E