1. DNA - The Molecular Basis of Inheritance

2. Important Early Discoveries
Fred Griffith (1928) – Experiments with pneumonia and bacterial transformation determined that there is a molecule that controls inheritance.
Oswald T. Avery (1944) - Transformation experiment determined that DNA was the genetic material responsible for Griffith’s results (not RNA).
Hershey-Chase Experiments (1952) – discovered that DNA from viruses can program bacteria to make new viruses.
Erwin Chargaff (1947) – noted that the the amount of A=T and G=C and an overall regularity in the amounts of A,T,C and G within species.

3. Frederick Griffith’s Transformation Experiment
The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928
Griffith worked with two strains of a bacterium, a pathogenic “S” strain and a harmless “R” strain
When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic
He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNA
Living S cells
(control)
Living R cells
Heat-killed
S cells (control)
Mixture of heat-killed
S cells and living R cells
Mouse dies
are found in
blood sample
Mouse healthy
RESULTS

4. Oswald T. Avery’s Transformation Experiment
In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA
Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria
Many biologists remained skeptical, mainly because little was known about DNA

5. Life Cycle Of Virulent T2 Phage

6. Hershey-Chase Bacteriophage Experiment
In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2
To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection
32P is discovered within the bacteria and progeny phages, whereas 35S is not found within the bacteria but released with phage ghosts.
They concluded that the injected DNA of the phage provides the genetic information
Bacterial cell
Phage
DNA
Radioactive
protein
Empty
protein shell
Radioactivity
(phage protein)
in liquid
Batch 1:
Sulfur (35S)
Centrifuge
Pellet (bacterial
cells and contents)
Pellet
(phage DNA)
in pellet
Batch 2:
Phosphorus (32P)

7. Additional Evidence That DNA Is the Genetic Material
In 1947, Erwin Chargaff reported that DNA composition varies from one species to the next
This evidence of diversity made DNA a more credible candidate for the genetic material
By the 1950s, it was already known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group
Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helical
The X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases
The width suggested that the DNA molecule was made up of two strands, forming a double helix

8. James D. Watson & Francis H. Crick
In 1953 presented the double helix model of DNA
Two primary sources of information:
1. Chargaff Rule: #A#T and #G#C. “A strange but possibly meaningless phenomenon”.
2. X-ray diffraction studies of Rosalind Franklin & Maurice H. F. Wilkins

9. DNA Structure
Conclusion-DNA is a helical structure with distinctive regularities, 0.34 nm & 3.4 nm.

10. 1962: Nobel Prize in Physiology and Medicine
Watson, J.D. and F.H. Crick, “Molecular Structure of Nucleic Acids: A Structure for Deoxynucleic Acids”. Nature 171 (1953), p. 738.
James D.
Watson
Francis H.
Crick
Maurice H. F.
Wilkins
What about?
Rosalind Franklin

11. The Structure of DNA
DNA is composed of four nucleotides, each containing: adenine, cytosine, thymine, or guanine.
The amounts of A = T, G = C, and purines = pyrimidines [Chargaff’s Rule].
DNA is a double-stranded helix with antiparallel strands [Watson and Crick].
Nucleotides in each strand are linked by 5’-3’ phosphodiester bonds
Bases on opposite strands are linked by hydrogen bonding: A with T, and G with C.

12. The Basic Principle: Base Pairing to a Template Strand
The relationship between structure and function is manifest in the double helix
Since the two strands of DNA are complementary each strand acts as a template for building a new strand in replication
In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules
5 end
3 end
Hydrogen bond
0.34 nm
3.4 nm
1 nm

13. DNA replication
The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules
(a) The parent molecule has two complementary strands of DNA Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.
(b) The first step in replication is separation of the two DNA strands.
(c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.
(d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands Each “daughter” DNA molecule consists of one parental strand and one new strand. A C T G T T G A C A C T G T G A C A C T G A T G A C A C T G T G A C A C T G T G A C G C A T T A C G

14. DNA Replication DNA must replicate during each cell division
3 alternative models for DNA replication were hypothesized:
Semiconservative replication
Conservative replication
Dispersive replication
Semi-conservative
Conservative
Dispersive

15. Meselson-Stahl Experiments
Labeled the nucleotides of old strands with a heavy isotope of nitrogen (15N), new nucleotides were indicated by a lighter isotope (14N).
The first replication in the 14N medium produced a band of hybrid (15N-14N) DNA, eliminating the conservative model.
A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model.
Bacteria
cultured in
medium
containing
15N
DNA sample
centrifuged
after 20 min
(after first
replication)
after 40 min
(after second
transferred to
14N
Less
dense
More
Conservative
model
First replication
Semiconservative
Second replication
Dispersive

16. DNA Replication is “Semi-conservative”
Each 2-stranded daughter molecule is only half new
One original strand was used as a template to make the new strand

17. DNA Replication
The copying of DNA is remarkable in its speed and accuracy
Involves unwinding the double helix and synthesizing two new strands.
More than a dozen enzymes and other proteins participate in DNA replication
The replication of a DNA molecule begins at special sites called origins of replication, where the two strands are separated

18. Origins of Replication
A eukaryotic chromosome may have hundreds or even thousands of replication origins
Replication begins at specific sites
where the two parental strands
separate and form replication
bubbles.
The bubbles expand laterally, as
DNA replication proceeds in both
directions.
Eventually, the replication
bubbles fuse, and synthesis of
the daughter strands is
complete. 1 2 3
Origin of replication
Bubble
Parental (template) strand
Daughter (new) strand
Replication fork
Two daughter DNA molecules
In eukaryotes, DNA replication begins at many sites along the giant
DNA molecule of each chromosome.
In this micrograph, three replication
bubbles are visible along the DNA of
a cultured Chinese hamster cell (TEM).
(b)
(a)
0.25 µm

19. Mechanism of DNA Replication
DNA polymerase I degrades the RNA primer and replaces it with DNA
DNA polymerase III adds nucleotides to primer
DNA replication is catalyzed by DNA polymerase III which needs an RNA primer
DNA polymerase III cannot initiate the synthesis of a polynucleotide, they can only add nucleotides to the 3 end
The initial nucleotide strand is an RNA primer
RNA primase synthesizes primer on DNA strand
DNA polymerase adds nucleotides to the 3’ end of the growing strand

20. Mechanism of DNA Replication
Nucleotides are added by complementary base pairing with the template strand
DNA always reads from 5’ end to 3’ end for transcription replication
During replication, new nucleotides are added to the free 3’ hydroxyl on the growing strand
The nucleotides (deoxyribonucleoside triphosphates) are hydrolyzed as added, releasing energy for DNA synthesis.
The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells
New strand
5¢ end
Phosphate
Base
Sugar
Template strand
3¢ end
Nucleoside
triphosphate
DNA polymerase
Pyrophosphate

21. The Mechanism of DNA Replication
DNA synthesis on the leading strand is continuous
Only one primer is needed for synthesis of the leading strand
The lagging strand grows the same general direction as the leading strand (in the same direction as the Replication Fork). However, DNA is made in the 5’-to-3’ direction
Therefore, DNA synthesis on the lagging strand is discontinuous
For synthesis of the lagging strand, each fragment (Okazaki) must be primed separately, then DNA fragments are sythesized and subsequently ligated together
Parental DNA 5¢ 3¢
Leading strand
Okazaki
fragments
Lagging strand
DNA pol III
Template
strand
DNA ligase
Overall direction of replication

22. Mechanism of DNA Replication
Many proteins assist in DNA replication
DNA helicases unwind the double helix, the template strands are stabilized by other proteins
Single-stranded DNA binding proteins make the template available
RNA primase catalyzes the synthesis of short RNA primers, to which nucleotides are added.
DNA polymerase III extends the strand in the 5’-to-3’ direction
DNA polymerase I degrades the RNA primer and replaces it with DNA
DNA ligase joins the DNA fragments into a continuous daughter strand 5¢ 3¢
Parental DNA
Overall direction of replication
DNA pol III
Replication fork
Leading
strand
DNA ligase
Primase
OVERVIEW
Primer
DNA pol I
Lagging
Origin of replication

23. Enzymes in DNA replication
Helicase unwinds
parental double helix
Binding proteins
stabilize separate
strands
Primase adds
short primer
to template strand
DNA polymerase III
binds nucleotides
to form new strands
DNA polymerase I (Exonuclease) removes RNA primer and inserts the correct bases
Ligase joins Okazaki
fragments and seals
other nicks in sugar-phosphate backbone

24. Replication Helicase protein binds to DNA sequences called3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’
Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
Binding proteins prevent single strands from rewinding.
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.

25. Replication DNA polymerase III enzyme adds DNA nucleotides
Overall direction
of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’
DNA polymerase III enzyme adds DNA nucleotides
to the RNA primer.

26. Replication DNA polymerase proofreads bases added and
Overall direction
of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’
DNA polymerase proofreads bases added and
replaces incorrect nucleotides.

27. Replication Leading strand synthesis continues in a
Overall direction
of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’
Leading strand synthesis continues in a
5’ to 3’ direction.

28. Replication Leading strand synthesis continues in a
Overall direction
of replication 3’ 3’ 5’ 5’
Okazaki fragment 3’ 3’ 5’ 5’ 3’ 5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

29. Replication Leading strand synthesis continues in a
Overall direction
of replication 3’ 3’ 5’ 5’
Okazaki fragment 3’ 3’ 5’ 5’ 3’ 5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

30. Replication Leading strand synthesis continues in a
Overall direction
of replication 3’ 3’ 5’ 5’
Okazaki fragment 3’ 5’ 3’ 5’ 3’ 5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

31. Replication Leading strand synthesis continues in a3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

32. Replication Leading strand synthesis continues in a3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’
Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis produces 5’ to 3’ DNA
segments called Okazaki fragments.

33. Replication 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 3’ 5’ 5’
Exonuclease activity of DNA polymerase I removes RNA primers.

34. Replication Polymerase activity of DNA polymerase I fills the gaps.3’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’
Polymerase activity of DNA polymerase I fills the gaps.
Ligase forms bonds between sugar-phosphate backbone.

35. Replication Fork Overview5¢ 3¢
Parental DNA
Overall direction of replication
DNA pol III
Replication fork
Leading
strand
DNA ligase
Primase
OVERVIEW
Primer
DNA pol I
Lagging
Origin of replication

36. Other Proteins That Assist DNA Replication
Helicase, topoisomerase, single-strand binding protein are all proteins that assist DNA replication

37. Proofreading DNA must be faithfully replicated…but mistakes occur
DNA polymerase (DNA pol) inserts the wrong nucleotide base in 1/10,000 bases
DNA pol has a proofreading capability and can correct errors
Mismatch repair: ‘wrong’ inserted base can be removed
Excision repair: DNA may be damaged by chemicals, radiation, etc. Mechanism to cut out and replace with correct bases

38. Mutations
A mismatching of base pairs, can occur at a rate of 1 per 100,000 bases.
DNA polymerase proofreads and repairs accidental mismatched pairs.
Chances of a mutation occurring at any one gene is over 1 in 10,000,000,000 (billion)
Because the human genome is so large, even at this rate, mutations add up. Each of us probably inherited 3-4 mutations!

39. Proofreading and Repairing DNA
DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides
In mismatch repair of DNA, repair enzymes correct errors in base pairing
In nucleotide excision DNA repair nucleases cut out and replace damaged stretches of DNA
Nuclease
DNA
polymerase
ligase
A thymine dimer
distorts the DNA molecule. 1
A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed. 2
Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides. 3
DNA ligase seals the
Free end of the new DNA
To the old DNA, making the
strand complete. 4

40. DNA repair

41. Accuracy of DNA Replication
The chromosome of E. coli bacteria contains about 5 million bases pairs
Capable of copying this DNA in less than an hour
The 46 chromosomes of a human cell contain about 6 BILLION base pairs of DNA!!
Printed one letter (A,C,T,G) at a time…would fill up over 900 volumes of Campbell.
Takes a cell a few hours to copy this DNA
With amazing accuracy – an average of 1 per billion nucleotides

42. Replicating the Ends of DNA Molecules
The ends of eukaryotic chromosomal DNA get shorter with each round of replication
End of parental
DNA strands
Leading strand
Lagging strand
Last fragment
Previous fragment
RNA primer
Removal of primers and
replacement with DNA
where a 3 end is available
Primer removed but
cannot be replaced
with DNA because
no 3 end available
for DNA polymerase
Second round
of replication
New leading strand
New lagging strand 5
Further rounds
Shorter and shorter
daughter molecules 5 3

43. Telomeres
Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences, called telomeres, that postpone the erosion of genes near the ends of DNA molecules
1 µm

44. Telomerases
If the chromosomes of germ cells became shorter in every cell cycle essential genes would eventually be missing from the gametes they produce
An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells