2. Evidence That DNA Can Transform Bacteria
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

3. Mixture of heat-killed S cells and living R cells Living S cells
(control)
Living R cells
(control)
Heat-killed
S cells (control)
RESULTS
Mouse dies
Mouse healthy
Mouse healthy
Mouse dies
Living S cells
are found in
blood sample

4. 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. Evidence That Viral DNA Can Program Cells
More evidence for DNA as the genetic material came from studies of a virus that infects bacteria
Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research
Animation: Phage T2 Reproductive Cycle

6. LE 16-3
Phage
head
Tail
Tail fiber
DNA
100 nm
Bacterial
cell

7. Animation: Hershey-Chase 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
They concluded that the injected DNA of the phage provides the genetic information
Animation: Hershey-Chase Experiment

8. LE 16-4 Empty Radioactive protein shell Radioactivity Phage protein
in liquid
Phage
Bacterial cell
Batch 1:
Sulfur (35S)
DNA
Phage
DNA
Centrifuge
Radioactive
DNA
Pellet (bacterial
cells and contents)
Batch 2:
Phosphorus (32P)
Centrifuge
Radioactivity
(phage DNA)
in pellet
Pellet

9. 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
Animation: DNA and RNA Structure

10. LE 16-5 Sugar–phosphate backbone Nitrogenous bases 5 end Thymine (T)
Adenine (A)
Cytosine (C)
Phosphate
DNA nucleotide
Sugar (deoxyribose)
3 end
Guanine (G)

11. Building a Structural Model of DNA: Scientific Inquiry
After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure accounts for its role
Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure
Franklin produced a picture of the DNA molecule using this technique

12. Franklin’s X-ray diffraction photograph of DNA
LE 16-6
Rosalind Franklin
Franklin’s X-ray diffraction
photograph of DNA

13. Animation: DNA Double Helix
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
Animation: DNA Double Helix

14. LE 16-7 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end
Key features of DNA structure
Partial chemical structure
Space-filling model

15. Watson and Crick built models of a double helix to conform to the X-rays and chemistry of DNA
Franklin had concluded that there were two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior
At first, Watson and Crick thought the bases paired like with like (A with A, and so on), but such pairings did not result in a uniform width
Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X-ray

16. Purine + purine: too wide
LE 16-UN298
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width
consistent with X-ray data

17. Watson and Crick reasoned that the pairing was more specific, dictated by the base structures
They determined that adenine paired only with thymine, and guanine paired only with cytosine

18. LE 16-8 Sugar Sugar Adenine (A) Thymine (T) Sugar Sugar Guanine (G)
Cytosine (C)

19. Concept 16.2: Many proteins work together in DNA replication and repair
The relationship between structure and function is manifest in the double helix
Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material

20. The Basic Principle: Base Pairing to a Template Strand
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
Animation: DNA Replication Overview

21. 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.

22. 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.
The first step in replication is separation of the two DNA strands.

23. LE 16-9_3 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.
The first step in replication is separation of the two DNA strands.
Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

24. LE 16-9_4 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.
The first step in replication is separation of the two DNA strands.
Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.
The nucleotides are connected to form the sugar-phosphate back-
bones of the new strands. Each “daughter” DNA
molecule consists of one parental strand and one new strand.

25. Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand
Competing models were the conservative model and the dispersive model

26. LE 16-10 First Second replication replication Parent cell
Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix.
Semiconservative model. The two strands of the parental
molecule
separate, and each functions as a template for synthesis of a new, comple-mentary strand.
Dispersive model. Each strand of both daughter molecules contains
a mixture of
old and newly synthesized
DNA.

27. Experiments by Meselson and Stahl supported the semiconservative model
They labeled the nucleotides of the old strands with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope
The first replication produced a band of hybrid DNA, eliminating the conservative model
A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model

28. LE 16-11 Bacteria cultured in medium containing 15N Bacteria
transferred to
medium
containing
14N
DNA sample
centrifuged
after 20 min
(after first
replication)
DNA sample
centrifuged
after 40 min
(after second
replication)
Less
dense
More
dense
First replication
Second replication
Conservative
model
Semiconservative
model
Dispersive
model

29. DNA Replication: A Closer Look
The copying of DNA is remarkable in its speed and accuracy
More than a dozen enzymes and other proteins participate in DNA replication

30. Getting Started: Origins of Replication
Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble”
A eukaryotic chromosome may have hundreds or even thousands of origins of replication
Replication proceeds in both directions from each origin, until the entire molecule is copied
At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating

31. LE 16-12 Parental (template) strand 0.25 µm Origin of replication
Daughter (new) strand
Bubble
Replication fork
Two daughter DNA molecules
In eukaryotes, DNA replication begins at may 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).

32. Animation: Origins of Replication

33. Elongating a New DNA Strand
Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork
Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate
The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells

34. DNA polymerase Pyrophosphate Nucleoside triphosphate
New strand
Template strand
5¢ end
3¢ end
5¢ end
3¢ end
Sugar
Base
Phosphate
DNA polymerase
3¢ end
3¢ end
Pyrophosphate
Nucleoside
triphosphate
5¢ end
5¢ end

35. Antiparallel Elongation
The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication
DNA polymerases add nucleotides only to the free 3end of a growing strand; therefore, a new DNA strand can elongate only in the 5 to 3direction

36. Along one template strand of DNA, called the leading strand, DNA polymerase can synthesize a complementary strand continuously, moving toward the replication fork
To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork
The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase

37. LE 16-14 3¢ 5¢ Parental DNA Leading strand 5¢ 3¢ Okazaki fragments
Lagging strand 3¢ 5¢
DNA pol III
Template
strand
Leading strand
Lagging strand
Template
strand
DNA ligase
Overall direction of replication

38. Animation: Leading Strand

39. Priming DNA Synthesis
DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end
The initial nucleotide strand is a short one called an RNA or DNA primer
An enzyme called primase can start an RNA chain from scratch
Only one primer is needed to synthesize the leading strand, but for the lagging strand each Okazaki fragment must be primed separately

40. LE 16-15_1 Primase joins RNA nucleotides into a primer. 3¢ 5¢ 5¢ 3¢
Template
strand
Overall direction of replication

41. LE 16-15_2 Primase joins RNA nucleotides into a primer. 3¢ 5¢ 5¢ 3¢
Template
strand
DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment. 3¢
RNA primer 5¢ 3¢ 5¢
Overall direction of replication

42. LE 16-15_3 Primase joins RNA nucleotides into a primer. 3¢ 5¢ 5¢ 3¢
Template
strand
DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment. 3¢
RNA primer 3¢ 5¢ 5¢
After reaching the
next RNA primer (not
shown), DNA pol III
falls off.
Okazaki
fragment 3¢ 3¢ 5¢ 5¢
Overall direction of replication

43. LE 16-15_4 Primase joins RNA nucleotides into a primer. 3¢ 5¢ 5¢ 3¢
Template
strand
DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment. 3¢
RNA primer 3¢ 5¢ 5¢
After reaching the
next RNA primer (not
shown), DNA pol III
falls off.
Okazaki
fragment 3¢ 3¢ 5¢ 5¢
After the second fragment is
primed, DNA pol III adds DNA
nucleotides until it reaches the
first primer and falls off. 5¢ 3¢ 3¢ 5¢
Overall direction of replication

44. LE 16-15_5 Primase joins RNA nucleotides into a primer. 3¢ 5¢ 5¢ 3¢
Template
strand
DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment. 3¢
RNA primer 3¢ 5¢ 5¢
After reaching the
next RNA primer (not
shown), DNA pol III
falls off.
Okazaki
fragment 3¢ 3¢ 5¢ 5¢
After the second fragment is
primed, DNA pol III adds DNA
nucleotides until it reaches the
first primer and falls off. 5¢ 3¢ 3¢ 5¢
DNA pol I replaces
the RNA with DNA,
adding to the 3¢ end
of fragment 2. 5¢ 3¢ 3¢ 5¢
Overall direction of replication

45. LE 16-15_6 Primase joins RNA nucleotides into a primer. 3¢ 5¢ 5¢ 3¢
Template
strand
DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment. 3¢
RNA primer 3¢ 5¢ 5¢
After reaching the
next RNA primer (not
shown), DNA pol III
falls off.
Okazaki
fragment 3¢ 3¢ 5¢ 5¢
After the second fragment is
primed, DNA pol III adds DNA
nucleotides until it reaches the
first primer and falls off. 5¢ 3¢ 3¢ 5¢
DNA pol I replaces
the RNA with DNA,
adding to the 3¢ end
of fragment 2. 5¢ 3¢ 3¢ 5¢
DNA ligase forms a
bond between the newest
DNA and the adjacent DNA
of fragment 1.
The lagging
strand in the region
is now complete. 5¢ 3¢ 3¢ 5¢
Overall direction of replication

46. Animation: Lagging Strand

47. Other Proteins That Assist DNA Replication
Helicase untwists the double helix and separates the template DNA strands at the replication fork
Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template
Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands

48. Primase synthesizes an RNA primer at the 5 ends of the leading strand and the Okazaki fragments
DNA pol III continuously synthesizes the leading strand and elongates Okazaki fragments
DNA pol I removes primer from the 5 ends of the leading strand and Okazaki fragments, replacing primer with DNA and adding to adjacent 3 ends
DNA ligase joins the 3 end of the DNA that replaces the primer to the rest of the leading strand and also joins the lagging strand fragments

49. LE 16-16 Leading strand Lagging strand Origin of replication Lagging
Overall direction of replication
Leading
strand
Lagging
strand
Origin of replication
Lagging
strand
Leading
strand
OVERVIEW
DNA pol III
Leading
strand
DNA ligase
Replication fork 5¢
DNA pol I 3¢
Primase
Parental DNA
DNA pol III
Lagging
strand
Primer 3¢ 5¢

50. Animation: DNA Replication Review

51. The DNA Replication Machine as a Stationary Complex
The proteins that participate in DNA replication form a large complex, a DNA replication “machine”
The DNA replication machine is probably stationary during the replication process
Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA and “extrude” newly made daughter DNA molecules

52. 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 repair, enzymes cut out and replace damaged stretches of DNA

53. LE 16-17 A thymine dimer distorts the DNA molecule.
A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
Nuclease
Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA
polymerase
DNA
ligase
DNA ligase seals the
free end of the new DNA
to the old DNA, making the
strand complete.

54. Replicating the Ends of DNA Molecules
Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes
The usual replication machinery provides no way to complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules

55. LE 16-18 5¢ End of parental DNA strands Leading strand Lagging strand3¢
Last fragment
Previous fragment
RNA primer
Lagging strand 5¢ 3¢
Primer removed but
cannot be replaced
with DNA because
no 3¢ end available
for DNA polymerase
Removal of primers and
replacement with DNA
where a 3¢ end is available 5¢ 3¢
Second round
of replication 5¢
New leading strand 3¢
New leading strand 5¢ 3¢
Further rounds
of replication
Shorter and shorter
daughter molecules

56. Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences called telomeres
Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules

57. LE 16-19
1 µm

58. If 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