DNA The Carrier of Genetic Information SL and HL

12 DNA: The Carrier of Genetic Information
After the rediscovery of Mendel’s principles in 1900, geneticists con-

ducted experiments to learn how genes are arranged in chromosomes
and how they are transmitted from generation to generation. How- key concepts
ever, basic questions remained unanswered through most of the first
12.1 In the 1920s, evidence began to accumulate that DNA is
half of the 20th century. What are genes made of? How do genes
the hereditary material.
work? The studies of inheritance patterns described in Chapter 11
did not answer these questions, but they did provide a foundation 12.2 The DNA molecule consists of two strands that wrap
around each other to form a double helix; the order
of knowledge that enabled scientists to make predictions about the
of its building blocks provides for the storage of
molecular (chemical) nature of genes and how genes function.
genetic information. The DNA building blocks consist of
Scientists generally agreed that genes would have to store four different nucleotide subunits, designated T, C, A,
information in a stable form that could be accurately copied and G. The pairing of nucleotide subunits occurs based
and passed unchanged from generation to generation. It was on precise pairing rules: T pairs with A, and C pairs
also known that a gene could sometimes convert to a different with G.
and stable form that would be transmitted unchanged to future 12.3 DNA replication involves the action of many different
generations. These genetic changes were called mutations. proteins. It results in two identical double-stranded
As the science of genetics was developing, biochemists were DNA molecules and provides the molecular mechanism
working to correlate the known properties of genes with the nature for passing genetic information from one generation
of various biological molecules. What kind of molecule could store to the next.
information? How could that information be retrieved and used
to direct cell functions? What kind of molecule could be relatively
stable but have the capacity to change, resulting in a mutation,
under certain conditions?
As they learned more about the central role of proteins in virtually
photo: Electron micrograph of DNA replication.
every aspect of cell structure and metabolism, some scientists con- During replication, two DNA molecules are synthesized from
sidered these complex and diverse molecules the prime candidates the original parent molecule. Replication is occurring at the
for the genetic material. However, protein did not turn out to be the Y-shaped structure, which is called a replication fork. Dr. Gopal Murti/
molecule that governs inheritance. In this chapter we discuss how Visuals Unlimited
253
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researchers discovered that deoxyribonucleic acid (DNA), a nucleic Because neither the heat-killed S strain nor the living
acid once thought unremarkable, is the molecular basis of inheri- R strain could be converted to the living virulent form when
tance. We explore the unique features of DNA, including its structure injected by itself in the control experiments, it seemed that
and replication (see photograph), that enable it to carry out this role. something in the heat-killed cells converted the avirulent
cells to the lethal form. This type of permanent genetic
change in which the properties of one strain of dead cells
12.1 Evidence of Dna as the are conferred on a different strain of living cells is called
transformation. Scientists hypothesized that some chemi-
Hereditary Material cal substance (a transforming principle, or factor) was trans-
ferred from the dead bacteria to the living cells and caused
learning objectives
transformation.
1 Summarize the evidence that accumulated during the In 1944, Oswald T. Avery and his colleagues Colin M.
1940s and early 1950s demonstrating that DNA is the MacLeod and Maclyn McCarty chemically identified Griffith’s
genetic material. transforming factor as DNA. They did so through a series of
2 State the questions that these classic experiments careful experiments in which they lysed (split open) S cells
addressed: Griffith’s transformation experiment, Avery’s and separated the cell contents into several fractions: lipids,
contribution to Griffith’s work, and the Hershey–Chase proteins, polysaccharides, and nucleic acids (DNA and RNA).
experiments. They tested each fraction to see if it could transform living
R cells into S cells. The experiments using lipids, polysaccha-
During the 1930s and early 1940s, most geneticists paid rides, and proteins did not cause transformation. However,
little attention to DNA, convinced that the genetic material when Avery treated living R cells with nucleic acids extracted
must be protein. Given the accumulating evidence that genes from S cells, the R cells were transformed into S cells.
control production of proteins (discussed in Chapter 13), it Although today scientists consider these results to be the
certainly seemed likely that genes themselves must also be first definitive demonstration that DNA is the genetic material,
proteins. Scientists knew proteins consisted of more than not all scientists of the time were convinced. Many thought
20 different kinds of amino acids in many different combi- that the findings might apply only to bacteria and might not
nations, which conferred unique properties on each type of have any relevance for the genetics of eukaryotes. During the
protein. Given their complexity and diversity compared with next few years, new evidence accumulated that the haploid
other molecules, proteins seemed to be the “stuff ” of which nuclei of pollen grains and gametes such as sperm contain
genes are made. only half the amount of DNA found in diploid somatic cells
In contrast, scientists had established that DNA and other of the same species. (Somatic cells are body cells and never
nucleic acids were made of only four nucleotides, and what become gametes.) Because scientists generally accepted that
was known about their arrangement made them relatively genes are located on chromosomes, these findings correlat-
uninteresting to most researchers. For this reason, several ing DNA content with chromosome number provided strong
early clues to the role of DNA were not widely noticed. circumstantial evidence for DNA’s importance in eukaryotic
inheritance.
DNA is the transforming
factor in bacteria DNA is the genetic material
One of these clues had its origin in 1928, when Frederick
in certain viruses
Griffith, a British medical officer, made a curious observation In 1952, geneticists Alfred Hershey and Martha Chase per-
concerning two strains of pneumococcus bacteria (FIG. 12-1). formed a series of elegant experiments on the reproduction
A smooth (S) strain, named for its formation of smooth colo- of viruses that infect bacteria, known as bacteriophages or
nies on a solid growth medium, exhibited virulence, the ability phages (discussed in Chapter 24). When they planned their
to cause disease and often death, in its host. When living cells experiments, they knew that phages reproduce inside a bacte-
of this strain were injected into mice, the animals contracted rial cell, eventually causing the cell to break open and release
pneumonia and died. Not surprisingly, the injected animals large numbers of new viruses. Because electron microscopic
survived if the cells were first killed with heat. A related rough studies had shown that only part of an infecting phage actu-
(R) strain of bacteria, which forms colonies with a rough sur- ally enters the cell, they reasoned that the genetic material
face, exhibited avirulence, or inability to produce pathogenic should be included in that portion (FIG. 12-2).
effects; mice injected with either living or heat-killed cells of As shown in FIGURE 12-3, they labeled the viral protein of
this strain survived. However, when Griffith injected mice one sample of phages with 35S, a radioactive isotope of sulfur,
with a mixture of heat-killed virulent S cells and live avirulent and the viral DNA of a second sample with 32P, a radioac-
R cells, a high proportion of the mice died. Griffith then iso- tive isotope of phosphorus. Recall from Chapter 3 that pro-
lated living S cells from the dead mice. teins contain sulfur as part of the amino acids cysteine and
254 / Chapter 12
Key Experiment
Can a genetic trait be transmitted from one bacterial strain to another?
hypothesis : The ability of pneumococ- Experiment 1 Experiment 2 Experiment 3 Experiment 4

cus bacteria to cause disease can be transmit-
ted from the virulent strain (smooth, or S cells)
to the avirulent strain (rough, or R cells).
experiment : Griffith performed
four experiments on mice, using the two
strains of pneumococci: (1) injection of
mice with live rough cells, (2) injection
with live smooth cells, (3) injection with
heat-killed smooth cells, and (4) injection R cells S cells Heat-killed R cells and heat-killed
with both live rough cells and heat-killed injected. injected. S cells injected. S cells injected.
smooth cells.
results and conclusion :
Although neither the rough strain nor the
heat-killed smooth strain could kill a mouse,
a combination of the two did. Autopsy
of the dead mouse showed the presence
of living S-strain pneumococci. These
results indicated that some substance in
the heat-killed S cells had transformed the
living R cells into a virulent form.
SOURCE: F. Griffith, “The Significance of
Pneumococcal Types,” Journal of Hygiene,
Vol. 27, No. 2 (1928): 113–159.
p r e d i c t What outcome would be Mouse lives. Mouse dies. Mouse lives. Mouse dies.
expected for experiment 3 if the heat-killed
S cells were treated with enzymes that
would specifically (1) digest the cell walls
of the heat-killed cells? (2) digest proteins in Figure 12-1 Griffith’s transformation experiments
the heat-killed cells? (3) break down nucleic Griffith was trying to develop a vaccine against pneumonia when he serendipitously discovered
acids in the heat-killed cells? the phenomenon of transformation.
methionine and that nucleic acids contain

phosphate groups. The phages in each sam-
ple attached to bacteria, and the researchers
shook the phages off by agitating the sample in
a blender. Then they centrifuged the samples.
In the sample in which they had labeled
Bacteriophage
the proteins with 35 S, they subsequently found
radioactivity in the supernatant, indicating
© Eye of Science/Science Source
that the protein had not entered the cells.

Phage genetic
material
In the sample in which they had labeled the
DNA with 32 P, they found radioactivity asso-
ciated with the bacterial cells (in the pellet):
Bacterial cell DNA had actually entered the cells. Hershey
(E. coli)
and Chase concluded that phages inject
0.5 mm their DNA into bacterial cells, leaving most
of their protein on the outside. This finding
Figure 12-2 Bacteriophages on a bacterial cell emphasized the significance of DNA in viral
This colorized TEM shows several phages attached to the bacterium Escherichia coli. reproduction, and many scientists saw it as an
Note the viral genetic material being injected into the bacterium. important demonstration of DNA’s role as the
hereditary material.
DNA: The Carrier of Genetic Information / 255
Key Experiment
Is DNA or protein the genetic material in bacterial viruses (phages)?
hypothesis : DNA is the genetic material in bacterial viruses.

experiment : Hershey and Chase produced phage populations with either radioactively labeled DNA or radioactively labeled protein. In both cases, they
infected bacteria with the phages and then determined whether DNA or protein was injected into bacterial cells to direct the formation of new viral particles.
1 Radioactively labeled 2 Cells agitated in blender and centrifuged. Bacteria, 3 Pellets and supernatants
phages infect bacterial which are heavier, settle into pellet. Phages and tested for radioactivity.
cells. phage parts, which are lighter, remain in supernatant.
35S
35S
Experiment A, phages Bacteria in No radioactively labeled

35S-labeled protein pellet do not protein entered cells.
with 35S-labeled protein
coat (green) in supernatant contain 35S label.
32P
32P
Bacteria in pellet Some newly assembled

Experiment B, phages contain 32P-labeled viruses have radioactively
with 32P-labeled DNA (red) DNA. labeled DNA.
results and conclusion : The researchers could separate phage protein coats labeled with the radioactive isotope 35 S from infected bacterial
cells without affecting viral reproduction. However, they could not separate viral DNA labeled with the radioactive isotope 32P from infected bacterial cells,
thus demonstrating that viral DNA enters the bacterial cells and is required for the synthesis of new viral particles. Thus, DNA is the genetic material in phages.
SOURCE: Hershey, A.D., and M. Chase, “Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage,” Journal of General
Physiology, Vol. 36, No. 1 (1952): 39–56.
Figure 12-3 The Hershey–Chase experiments

c o n n e c t Explain how this experiment reinforces the earlier findings of Avery, McLeod, and McCarty that genes are composed of nucleic acids.
checkpoint 12.1 • connect How did Hershey and Chase use their
knowledge of the chemical composition of proteins and
• c o n n e c t How did Griffith’s work provide a foundation nucleic acids to design their experiments establishing that
for the experiments of Avery and his colleagues pointing DNA is the genetic material in bacteriophages?
to DNA as the essential genetic material?
256 / Chapter 12
12.2 The Structure of Dna Nucleotides can be covalently linked
in any order to form long polymers
learning objectives
As discussed in Chapter 3, each DNA building block is a
3 Explain how nucleotide subunits link to form a single DNA nucleotide consisting of the pentose sugar deoxyribose, a phos-
strand. phate, and one of four nitrogenous bases (FIG. 12-4). It is conven-
4 Describe how the two strands of DNA are oriented with tional to number the atoms in a molecule using a system devised
respect to each other. by organic chemists. Accordingly, in nucleic acid chemistry the
5 State the base-pairing rules for DNA and describe how individual carbons in each sugar and each base are numbered.
complementary bases bind to each other. The carbons in a base are designated by numerals, but the carbons
in a sugar are distinguished from those in the base by prime sym-
Scientists did not generally accept DNA as the genetic mate- bols, such as 29. The nitrogenous base is attached to the 19 carbon
rial until 1953, when U.S. scientist James Watson and British of the sugar, and the phosphate is attached to the 59 carbon. The
scientist Francis Crick, both working in England, proposed bases include two purines, adenine (A) and g uanine (G), and
a model for its structure that had extraordinary explanatory two pyrimidines, thymine (T) and cytosine (C).
power. The story of how the structure of DNA was figured out The nucleotides are linked by covalent bonds to form an
is one of the most remarkable chapters in the history of modern alternating sugar–phosphate backbone. The 39 carbon of one
biology (TABLE 12-1). As you will see in the following discus- sugar is bonded to the 59 phosphate of the adjacent sugar to form
sion, scientists already knew a great deal about DNA’s physi-
cal and chemical properties when Watson and Crick became O
interested in the problem; in fact, Watson and Crick did not 5′ H3C H
N
conduct any experiments or gather any new data. Their all-
important contribution was to integrate all the available infor- O
H N O
mation into a model that demonstrated how the molecule can 5′ Thymine
O P O CH 2
both carry information for making proteins and serve as its O
4′ H H
O– H H 1′ N
own template (pattern or guide) for its duplication. H H
3′ 2′
N N
H
H
O N H
N
Table 12-1 Time Line of Selected Historical DNA Discoveries Adenine Nucleotide
O P O CH2
O
Date Discovery O– H H H H
N
H H
1871 Friedrich Miescher reports discovery of new sub- H
stance, nuclein, from cell nuclei. Nuclein is now known H N
to be a mixture of DNA, RNA, and proteins.
O H N O
1928 Frederick Griffith finds a substance in heat-killed bac- Cytosine
teria that “transforms” living bacteria. Phosphate O P O CH2
group O
1944 Oswald Avery, Colin MacLeod, and Maclyn O– H H O
McCarty chemically identify Griffith’s transforming
H H
principle as DNA. N H
H N
1949 Erwin Chargaff reports relationships among DNA
bases that provide a clue to the structure of DNA. O H
N N N
1952 Alfred Hershey and Martha Chase demonstrate that Phosphodiester O P O CH2 Guanine H
DNA, not protein, is involved in viral reproduction. linkage O
1952 Rosalind Franklin produces X-ray diffraction images O– H H
of DNA. H 3′ H
1953 James Watson and Francis Crick propose a model of Deoxyribose OH H
the structure of DNA; this contribution is widely consid- (sugar)
ered the start of a revolution in molecular biology that
continues to the present.
3′
1958 Matthew Meselson and Franklin Stahl demonstrate
that DNA replication is semiconservative.
Figure 12-4 The nucleotide subunits of DNA
1962 James Watson, Francis Crick, and Maurice Wilkins
are awarded the Nobel Prize in Physiology or Medicine A single strand of DNA consists of a backbone (superimposed on
for discoveries about the molecular structure of nucleic blue screen) of phosphate groups alternating with the sugar deoxy-
acids.* ribose (green). Phosphodiester linkages (pink) join sugars of adjacent
1969 Alfred Hershey is awarded the Nobel Prize in Physiol- nucleotides. Linked to the 19 carbon of each sugar is one of four
ogy or Medicine for discovering the replication mecha- nitrogenous bases (top to bottom): thymine, adenine, cytosine, and
nism and genetic structure of viruses. guanine. (The nucleotide containing the base adenine is highlighted
yellow.) Note the polarity of the polynucleotide chain, with the
* Rosalind Franklin could not share the prize because she was deceased. 59 end at the top of the figure and the 39 end at the bottom.
a 39, 59 phosphodiester linkage. The result is a polymer of indefi-
nite length, with the nucleotides linked in any order. Scientists
now know that most DNA molecules found in cells are millions
of bases long. Figure 12-4 also shows that a single polynucleotide
chain is directional. No matter how long the chain may be, one
end, the 59 end, has a 59 carbon attached to a phosphate, and the
other end, the 39 end, has a 39 carbon attached to a hydroxyl group.
By 1949, Erwin Chargaff and his colleagues at Columbia
University had determined the base composition of DNA from
several organisms and tissues. They found a simple relation-
ship among the bases that turned out to be an important clue
to the structure of DNA. Regardless of the source of the DNA,
in Chargaff ’s words the “ratios of purines to pyrimidines and
also of adenine to thymine and of guanine to cytosine were not
far from 1” (TABLE 12-2). In other words, in double-stranded
DNA molecules, the number of purines equals the number
of pyrimidines, the number of adenines equals the number of
thymines (A equals T), and the number of guanines equals the
number of cytosines (G equals C). These equalities are called
Chargaff ’s rules.
DNA is made of two polynucleotide
National Library of Medicine/Science Source

chains intertwined to form a double helix
Key information about the structure of DNA came from X-ray
diffraction studies on crystals of purified DNA, carried out
by British scientist Rosalind Franklin at King’s College from
1951 to 1953 (FIG. 12-5).
X-ray diffraction, a powerful method for determining
the 3-D structure of a molecule, can determine the distances
between the atoms of molecules arranged in a regular, repeating Figure 12-5 Rosalind Franklin
crystalline structure (FIG. 12-6). X-rays have such short wave- Franklin was a gifted scientist whose contributions helped
lengths that they can be scattered by the electrons surround- elucidate the double-helix structure of DNA.
ing the atoms in a molecule. Atoms with dense electron clouds
(such as phosphorus and oxygen) tend to deflect electrons more
strongly than atoms with lower atomic numbers. Exposing a
crystal to an intense beam of X-rays causes the regular arrange- has a type of helical structure, and three major types of regular,
ment of its atoms to diffract, or scatter, the X-rays in specific repeating patterns in the molecule (with the dimensions 0.34 nm,
ways. The pattern of diffracted X-rays appears on photographic 3.4 nm, and 2.0 nm) were evident. Franklin and c olleague Maurice
film as dark spots. Mathematical analysis of the pattern and dis- Wilkins had inferred from these patterns that the nucleotide
tances between the spots yields the precise distances between bases (which are flat molecules) are stacked like the rungs of a
atoms and their orientation within the molecules. ladder. Using this information, Watson and Crick began to build
Franklin had already produced X-ray crystallographic films scale models of the DNA components and then fit them together
of DNA patterns when Watson and Crick began to pursue the to correlate with the experimental data (FIG. 12-7).
problem of DNA structure. Her pictures clearly showed that DNA After several trials, they worked out a model that fit the
existing data (FIG. 12-8). The nucleotide chains conformed to
Table 12-2 Base Compositions in DNA from Selected Organisms the dimensions of the X-ray data only if each DNA molecule
consisted of two polynucleotide chains arranged in a coiled
Percentage of Dna Bases Ratios
double helix. In their model the sugar–phosphate backbones
SOURCE OF DNA A T G C A/T G/C of the two chains form the outside of the helix. The bases
E. coli 26.1 23.9 24.9 25.1 1.09 0.99 belonging to the two chains associate as pairs along the cen-
Yeast 31.3 32.9 18.7 17.1 0.95 1.09 tral axis of the helix. The reasons for the repeating patterns
Sea urchin sperm 32.5 31.8 17.5 18.2 1.02 0.96 of 0.34 nm and 3.4 nm measurements are readily apparent
Herring sperm 27.8 27.5 22.2 22.6 1.01 0.98 from the model: each pair of bases is exactly 0.34 nm from
Human liver 30.3 30.3 19.5 19.9 1.00 0.98 the adjacent pairs above and below. Because exactly 10 base
Corn (Zea mays) 25.6 25.3 24.5 24.6 1.01 1.00
pairs are present in each full turn of the helix, each turn con-
stitutes 3.4 nm of length. To fit the data, the two chains must
258 / Chapter 12
Research Method
why is it used? X-ray diffraction can be used to determine the

regular array of atoms in a crystalline sample of, for example, DNA.
Because each type of crystal has its own characteristic pattern, the three-
dimensional structure of the molecule being studied can be deduced.
how is it done?
1. Researchers direct a narrow beam of X-rays at a single crystal of
DNA. X-rays are diffracted (bent) at specific angles based on the
density of electrons of different atoms. Important clues about
DNA structure are provided by detailed mathematical analysis of
measurements of the spots, which are formed by X-rays hitting
the photographic plate
A. Barrington Brown/Science Source

Beam of X-rays
X-ray source DNA sample

X-rays
diffracted
by DNA
Figure 12-7 James Watson and Francis Crick
Photographic plate
shows atomic array Watson (left) and Crick (right) are shown with their model of
of DNA. DNA’s double helix.
2. X-ray diffraction image of DNA. This diagonal pattern of spots

stretching from 11 to 5 and from 1 to 7 (as on a clock face) Sugar–phosphate backbone
provides evidence for the helical structure of DNA. The elon-
gated horizontal patterns at the top and bottom indicate that
the purine and pyrimidine bases are stacked 0.34 nm apart
and are perpendicular to the axis of the DNA molecule.
Minor
groove
Dr. S. Dover, Division of Biomolecular Sciences, Kings College, London
Major
3.4
groove
nm
KEY
Hydrogen
Oxygen
Carbon 0.34 nm
Figure 12-6 How X-ray diffraction works
Atoms in base pairs
Phosphorus 2.0 nm
run in opposite directions; therefore, each end of the double
helix must have an exposed 59 phosphate on one strand and an Figure 12-8 A three-dimensional model of the DNA
exposed 39 hydroxyl group (—OH) on the other. Because the double helix
two strands run in opposite directions, they are antiparallel The measurements match those derived from X-ray diffraction images.
to each other (FIG. 12-9a).
Key Point
Base pairing and the sequence of bases in DNA provide a foundation for understanding both DNA
replication and the inheritance of genetic material.
H
H
H
G H C
H Adenine Thymine
H H H H
A H T
CH2 N H O C H
O H N
O C
–O P O C C O C
O P O–
O
H O N
CH2 C N H N C H
C H G H OH
O
H N C C N
CH2
O
–O O H O
P O
O P O–
O HO H O
H O
CH2 A T Deoxyribose Deoxyribose
H
O CH2
–O O
P O
O P O–
O
H O
CH2 G C Guanine Cytosine
H
H H
O CH2
–O P O O O H N H
H N
O P O– C
O C C C C
H O
CH2
T H A N
C N H N C H
CH2 O H OH
O N C
O C N
–O P O
O P O– N H O
O
H O
CH2 G C H O
H HO H
H Deoxyribose Deoxyribose
3′ 5′
(a) The two sugar–phosphate chains run in opposite (b) Hydrogen bonding between base pairs adenine (A) and
directions. This orientation permits the complementary thymine (T) (top) and guanine (G) and cytosine (C) (bottom).
bases to pair. The A:T pair has two hydrogen bonds; the G:C pair has three.
Figure 12-9 Base pairing and hydrogen bonding predict The two strands of the DNA double helix are
The two strands of a DNA double helix are hydrogen-bonded between the bases. held together by the hydrogen bonding between the paired
bases. Which would require more energy to separate the two
strands, a DNA molecule composed mostly of G:C base pairs
or a DNA molecule with mostly A:T base pairs?
260 / Chapter 12
In double-stranded DNA, hydrogen
bonds form between A and T and checkpoint 12.2
between G and C • What types of subunits make up a single strand of DNA?
Other features of the Watson and Crick model integrated criti- How are the subunits linked?
cal information about the chemical composition of DNA with • What is the structure of double-stranded DNA as
the X-ray diffraction data. The X-ray diffraction studies indi- determined by Watson and Crick?
cated that the double helix has a precise and constant width, • connect How do Chargaff’s rules relate to the structure
as shown by the 2.0 nm measurements. This finding is actu- of DNA?
ally consistent with Chargaff ’s rules. As Figure 12-4 shows,
each pyrimidine (cytosine or thymine) contains only one ring
of atoms, whereas each purine (guanine or adenine) contains
two rings. Study of the models made it clear to W atson and
12.3 DNA Replication
Crick that if each rung of the ladder contained one purine
learning objectives
and one pyrimidine, the width of the helix at each base pair
would be exactly 2.0 nm. By contrast, the combination of 6 Cite evidence from Meselson and Stahl’s experiment that
two purines (each of which is 1.2 nm wide) would be wider enabled scientists to differentiate between semiconserva-
than 2.0 nm and that of two pyrimidines would be narrower, tive replication of DNA and alternative models.
so the diameter would not be constant. Further examination 7 Summarize how DNA replicates and identify some unique
of the model showed that adenine can pair with thymine (and features of the process.
guanine with cytosine) in such a way that hydrogen bonds 8 Explain the complexities of DNA replication that make the
form between them; the opposite combinations, cytosine with process (a) bidirectional and (b) continuous in one strand
adenine and guanine with thymine, do not lead to favorable and discontinuous in the other.
hydrogen bonding. 9 Discuss how enzymes proofread and repair errors in DNA.
The nature of the hydrogen bonding between adenine 10 Define telomere and describe the possible connections
and thymine and between guanine and cytosine is shown in between telomerase and cell aging and between
FIGURE 12-9b. Two hydrogen bonds form between adenine telomerase and cancer.
and thymine, and three form between guanine and cyto-
sine. This concept of specific base pairing neatly explains Two immediately apparent and distinctive features of the
Chargaff ’s rules. The amount of cytosine must equal the Watson–Crick model made it seem plausible that DNA is the
amount of guanine because every cytosine in one chain must genetic material. We have already mentioned that the sequence
have a paired guanine in the other chain. Similarly, every of bases in DNA can carry coded information. The model also
adenine in the first chain must have a thymine in the sec- suggested a way in which the sequence of nucleotides in DNA
ond chain. The sequences of bases in the two chains exhibit could be precisely copied, a process known as DNA replication.
complementary base pairing; that is, the sequence of nucle- The connection between DNA replication and the behav-
otides in one chain dictates the complementary sequence of ior of chromosomes in mitosis was obvious to Watson and
nucleotides in the other. For example, if one strand has the Crick. A chromosome becomes duplicated so that it con-
sequence sists of two identical sister chromatids that later separate at
anaphase; the genetic material must be precisely duplicated
3 AGCTAC 5 and distributed to the daughter cells. They noted, in a clas-
sic and now famous understatement at the end of their first
brief paper, “It has not escaped our notice that the specific
the other strand has the complementary sequence pairing we have postulated immediately suggests a possible
copying mechanism for the genetic material.”
5 TCGATG 3
The model suggested that because the nucleotides pair
with each other in complementary fashion, each strand of
the DNA molecule could serve as a template for synthesiz-
The double-helix model strongly suggested that the ing the opposite strand. It would simply be necessary for the
sequence of bases in DNA provides for the storage of genetic hydrogen bonds between the two strands to break (recall from
information and that this sequence ultimately relates to the Chapter 2 that hydrogen bonds are relatively weak) and the
sequences of amino acids in proteins. Although restrictions two chains to separate. Each strand of the double helix could
limit how the bases on opposite strands pair with each other, then pair with new complementary nucleotides to replace
the number of possible linear sequences of bases in a strand its missing partner. The result would be two DNA double
is virtually unlimited. Because a DNA molecule in a cell helices, each identical to the original one and consisting of
can be millions of nucleotides long, it can store enormous one original strand from the parent molecule and one newly
amounts of information, usually consisting of hundreds of synthesized complementary strand. This type of information
genes. copying is called semiconservative replication (FIG. 12-10a).
Meselson and Stahl verified the other models, investigators had to distinguish between old
mechanism of semiconservative and newly synthesized strands of DNA.
One technique is to use a heavy isotope of nitrogen, 15 N
replication (ordinary nitrogen is 14 N ), to label the bases of the DNA
Although the semiconservative replication mechanism sug- strands, making them more dense. Using density gradient
gested by Watson and Crick was (and is) a simple and compel- centrifugation, scientists can separate large molecules such as
ling model, experimental proof was needed to establish that DNA on the basis of differences in their density (see Fig. 4-6).
DNA in fact replicates in that manner. Researchers first needed When DNA is mixed with a solution containing cesium chlo-
to rule out other possibilities. With conservative replication, ride (CsCl) and centrifuged at high speed, the solution forms a
both parent (or old) strands would remain together, and the density gradient in the centrifuge tube, ranging from a region
two newly synthesized strands would form a second double of lowest density at the top to one of highest density at the
helix (FIG. 12-10b). As a third hypothesis, the parental and newly bottom. During centrifugation, the DNA molecules migrate
synthesized strands might become randomly mixed during the to the region of the gradient identical to their own density.
replication process, that is, dispersive replication (FIG. 12-10c). In 1958, Matthew Meselson and Franklin Stahl at the
To discriminate among semiconservative replication and the California Institute of Technology grew the bacterium
Escherichia coli on a medium that contained 15 N in the form
of ammonium chloride (NH 4 Cl). The cells used the 15 N to
Parental DNA First generation Second generation synthesize bases, which then became incorporated into DNA
(FIG. 12-11). The resulting DNA molecules, which contained
heavy nitrogen, were extracted from some of the cells. When the
researchers subjected them to density gradient centrifugation,
they accumulated in the high-density region of the gradient. The
team transferred the rest of the bacteria (which also contained
15
N-labeled DNA) to a different growth medium in which the
NH 4 Cl contained the naturally abundant, lighter 14 N isotope
and then allowed them to undergo additional cell divisions.
Meselson and Stahl expected the newly synthesized DNA
(a) Hypothesis 1: Semiconservative replication
strands to be less dense because they incorporated bases con-
taining the lighter 14 N isotope. Indeed, double-stranded DNA
Parental DNA First generation Second generation
from cells isolated after one generation had an intermediate
density, indicating that they contained half as many 15 N atoms
as the “parent” DNA. This finding supported the semicon-
servative replication model, which predicted that each double
helix would contain a previously synthesized strand (heavy in
this case) and a newly synthesized strand (light in this case).
It was also consistent with the dispersive model, which would
also yield one class of molecules, all with intermediate density.
It was inconsistent with the conservative model, which pre-
(b) Hypothesis 2: Conservative replication dicted two classes of double-stranded molecules: those with
two heavy strands and those with two light strands.
Parental DNA First generation Second generation After another cycle of cell division in the medium with the
lighter 14 N isotope, two types of DNA appeared in the density
gradient, exactly as predicted by the semiconservative replica-
tion model. One consisted of DNA with a density intermedi-
ate between 15 N-labeled DNA and 14 N -labeled DNA, whereas
the other contained only DNA with a density of 14 N -labeled
DNA. This finding refuted the dispersive model, which pre-
dicted that all strands would have an intermediate density.
(c) Hypothesis 3: Dispersive replication

Semiconservative replication explains
Figure 12-10 Alternative models of DNA replication the perpetuation of mutations
The hypothesized arrangement of old (light blue) and newly
synthesized (dark blue) DNA strands after one and two The recognition that DNA could be copied by a semicon-
generations, according to (a) the semiconservative model, servative mechanism suggested how DNA could explain a
(b) the conservative model, and (c) the dispersive model. third essential characteristic of genetic material, the ability to
mutate. It was long known that mutations, or genetic changes,
262 / Chapter 12
Key Experiment When the double-helix model was proposed, it seemed
plausible that mutations could represent a change in the
What is the mechanism of DNA replication? sequence of bases in the DNA. You could predict that if DNA is
copied by a mechanism involving complementary base pairing,
hypothesis : Figure 12-10 depicts three hypotheses of DNA any change in the sequence of bases on one strand would pro-
replication and predicts the arrangement of old and newly synthesized
DNA strands after one or two generations according to each hypothesis.
duce a new sequence of complementary bases during the next
replication cycle. The new base sequence would then transfer
experiment : Meselson and Stahl grew bacteria (E. coli) in heavy
to daughter molecules by the same mechanism used to copy
nitrogen ( 15N) growth medium for many generations so that all the DNA
strands would be heavy. Then they transferred some of the cells to light the original genetic material as if no change had occurred.
nitrogen ( 14 N) medium so that the newly synthesized strands would For the example in FIGURE 12-12, an adenine base in one of
be light. They isolated DNA from bacterial cells after 20 minutes (one the DNA strands has been changed to guanine. This change
generation) and 40 minutes (two generations), and centrifuged it to could occur by a rare error in DNA replication or by one of
separate DNA into bands based on density.
several other known mechanisms. As discussed later, certain
14N
enzymes correct errors when they occur, but not all errors are
(light) 14N–15N 15N (heavy) corrected properly. By one estimate, the rate of uncorrected
DNA hybrid DNA DNA
errors that occur during DNA replication is equal to about one
nucleotide in a billion. When the DNA molecule containing
14N (light) 14N–15N 15N (heavy) an error replicates (left side of Figure 12-12), one of the strands
DNA hybrid DNA DNA gives rise to a molecule exactly like its parent strand; the other
In density gradient centrifugation, the concentration of CsCl (mutated) strand gives rise to a molecule with a new combina-
is highest at the bottom of the tube and lowest at the top. DNA tion of bases that is transmitted generation after generation.
molecules move to positions where their density equals that of
the CsCl solution in which they are centrifuged.
In density gradient centrifugation, the concentration of CsCl
results and : Based
DNA replication requires protein
is highest at theconclusion
bottom of the tube on theatobserved
and lowest the top. density
DNA of
the molecules
DNA molecules
moveintoeach generation,
positions whereMeselson and equals
their density Stahl concluded
that of “machinery”
thatthe
theCsCl
semiconservative modelthey
solution in which accurately predicts the mechanism of
are centrifuged.
DNA replication. Although semiconservative replication by base pairing
14N appears simple and straightforward, the actual process
(light) is highly regulated and requires a “replication machine”
DNA
14N–15N 14N–15N
14N
hybrid hybrid
DNA (light)
DNA
DNA A T
15N
14N–15N
T A
(heavy) 14N–15N
DNA hybrid hybrid G C
DNA DNA C G
Before transfer
15N One cell generation Two cell generations
14N 14
to (heavy) after transfer to N after transfer to 14N
DNA replication
DNA
The location of DNA molecules within the centrifuge tube can in which mutation
be determined occurs
Before transfer by UV
Oneoptics. DNA solutions absorb
cell generation Two cellstrongly
generations
at 260
to 14N nm. after transfer to 14N after transfer to 14N
A T A T
The location of DNA molecules within the centrifuge tube can T G Mutation T A
be determined by UV optics. DNA solutions absorb strongly G C G C
at 260 nm.
C G C G
SOURCE: Meselson, M., and F.W. Stahl, “The Replication of DNA in
Escherichia coli,” Proceedings of the National Academy of Sciences DNA replication
U.S.A., Vol. 44, No. 7 (1958): 671–682. in which mutation
is propagated
Figure 12-11 The Meselson–Stahl experiment A T A T A T A T

v i s u a l i z e Draw the positions of the second-generation DNA T A C G T A T A
molecules you would expect to see in the tube on the right (two G C G C G C G C
generations) if they were replicated by (1) the conservative replication
C G C G C G C G
hypothesis and (2) the dispersive replication hypothesis.
Figure 12-12 The perpetuation of a mutation

could arise in genes and then be transmitted faithfully to suc- The process of DNA replication can stabilize a mutation (outlined in
ceeding generations. For example, a mutation in the fruit fly red) so that it is transmitted to future generations.
(Drosophila melanogaster) produces vestigial wings.
containing many types of protein and enzyme molecules. Table 12-3 Proteins Involved in DNA Replication
Many essential features of DNA replication are common to
all organisms, although prokaryotes and eukaryotes differ Enzyme Function
somewhat because their DNA is organized differently. In most
Helicase Opens the double helix at replication forks by
bacterial cells, such as E. coli, all or most of the DNA is in the disrupting the hydrogen bonds that hold the two
form of a single, circular, double-stranded DNA molecule. In strands together.
contrast, each unreplicated eukaryotic chromosome contains
Single-strand Binds to single strands of DNA and prevents the
a single, linear, double-stranded molecule associated with at binding (SSB) helix from re-forming before it can be used as a
least as much protein (by mass) as DNA. protein template for replication.
The process of DNA replication requires a large number of
Topoisomerase Breaks one or both DNA strands, preventing
proteins with different functions, many of which are organized excessive coiling during replication, and then
as multimolecular “machines.” For example, in the unicellular rejoins them in a more relaxed configuration.
yeast Saccharomyces cerevisiae, which is considered a relatively
DNA Links nucleotide subunits to form a new DNA
“simple” eukaryote, 88 genes are involved in DNA replication! polymerase strand complementary to a DNA template.
DNA strands must be unwound during replication DNA primase Synthesizes short RNA primers on the lagging
strand. Begins replication of the leading strand.
DNA replication begins at specific sites on the DNA mole-
cule, called origins of replication, where small sections of the DNA ligase Links Okazaki fragments by joining the 39 end
double helix unwind. DNA helicases are helix-destabilizing of the new DNA fragment to the 59 end of the
adjoining DNA.
enzymes (several have been identified) that bind to DNA at
the origin of replication and break hydrogen bonds, thereby
separating the two strands (FIG. 12-13; TABLE 12-3). Both DNA replication, torsional strain from supercoiling (excessive twist-
strands replicate at the same time at the junction between the ing) occurs in another part of the DNA molecule. Enzymes
separated strands, which is a Y-shaped structure called called topoisomerases produce breaks in the DNA molecules
the replication fork (see chapter-opening photograph). Heli- and then rejoin the strands, relieving strain and effectively pre-
case travels along the helix, opening the double helix like a venting supercoiling and knot formation during replication.
zipper during movement of the replication fork. There are two ways that topoisomerases reduce supercoil-
Once DNA helicases separate the strands, single-strand ing. Some topoisomerases produce a temporary break in the
binding (SSB) proteins bind to single DNA strands and stabilize polynucleotide backbone of a single strand of DNA, pass that
them, which prevents the double helix from re-forming until the strand through the excessively twisted part, and then reseal the
strands are replicated. SSB proteins also prevent the hydrolysis break. Other topoisomerases break both DNA strands, pass
of the single-strand regions by other enzymes. (Nucleases, as we some of the helix between the cut ends, and then reseal the
discuss later in the chapter, are involved in DNA repair.) break. Regardless of their modes of action, topoisomerases
Watson and Crick recognized that in their double-helix give replicating DNA a more relaxed configuration.
model, the two DNA strands wrap around each other like the
strands of a rope. If you try to pull the strands apart, the rope DNA synthesis always proceeds in a 5 ′ → 3 ′ direction
must either rotate or twist into tighter coils. You could expect The enzymes that catalyze the linking of successive n
ucleotide
similar results when complementary DNA strands are sepa- subunits are called DNA polymerases. They add nucleo-
rated for replication. As the DNA strands unwind to open for tides only to the 39 end of a growing polynucleotide strand,
Single-strand Origin of replication

binding protein
Direction of
DNA unwinding
Topoisomerase
3′ 5′
5′ 3′
DNA helix
Direction of
DNA unwinding
Helicase
Figure 12-13 DNA strand separation during DNA replication

DNA strands are separated at an origin of replication by ATP-powered DNA helicase enzymes to create
a “replication bubble” with a Y-shaped replication fork at each end of the bubble. Single-strand binding
proteins bind to the unpaired strands to keep them from re-forming the helix. Unwinding the strands
twists and creates strain on the double helix ahead of the replication fork. Topoisomerase enzymes relieve
this strain by breaking, swiveling, and rejoining the paired strands.
264 / Chapter 12
and this strand must be paired with the DNA template strand DNA replication is discontinuous in one strand and
(FIG. 12-14). Nucleotides with three phosphate groups are sub- continuous in the other We mentioned earlier that the
strates for the polymerization reaction. As the nucleotides complementary DNA strands are antiparallel. DNA synthe-
become linked, two of the phosphates are removed. These reac- sis proceeds only in the 5′ → 3′ direction, which means that
tions are strongly exergonic and do not need additional energy. the strand being copied is being read in the 3′ → 5′ direction.
Because the polynucleotide chain is elongated by the linkage of Thus, it may seem necessary to copy one of the strands starting
the 59 phosphate group of the next nucleotide subunit to the at one end of the double helix and the other strand starting at
39 hydroxyl group of the sugar at the end of the existing strand, the opposite end. Some viruses replicate their DNA in this way,
the new strand of DNA always grows in the 5′ → 3′ direction. but this replication method is not workable in the extremely
Some DNA polymerases are very efficient in joining nucleo- long DNA molecules in eukaryotic chromosomes.
tides to the growing polypeptide chain. DNA Pol III, which is Instead, as previously mentioned, DNA replication begins
one of five DNA polymerases that have been identified in the at origins of replication, and both strands replicate at the same
bacterium E. coli, can join 1200 nucleotides per minute. time at a replication fork (see Fig. 12-15). The position of the
replication fork is constantly moving as replication proceeds.
DNA synthesis requires an RNA primer As mentioned, Two identical DNA polymerase molecules catalyze replica-
DNA polymerases add nucleotides only to the 39 end of an tion. One of these adds nucleotides to the 39 end of the new
existing polynucleotide strand. Then how is DNA synthesis ini- strand that is always growing toward the replication fork.
tiated once the two strands are separated? The answer is that first Because this strand is synthesized smoothly and continuously,
a short piece of RNA (5 to 14 nucleotides) called an RNA primer it is called the leading strand.
is synthesized at the point where replication begins (FIG. 12-15). A separate DNA polymerase molecule adds nucleotides to
RNA, or ribonucleic acid (see Chapters 3 and 13), is a the 39 end of the other new strand. Called the lagging strand,
nucleic acid polymer consisting of nucleotide subunits that it is always growing away from the replication fork. Only short
can associate by complementary base pairing with the single- pieces can be synthesized, because if the DNA polymerase
strand DNA template. The RNA primer is synthesized by DNA were to add continuously to the 39 end of that strand, it would
primase, an enzyme that starts a new strand of RNA opposite need to move far from the replication fork. These 100- to
a short stretch of the DNA template strand. After a few nucleo- 2000-nucleotide pieces are called Okazaki fragments after
tides have been added, DNA polymerase displaces the primase their discoverer, Japanese molecular biologist Reiji Okazaki.
and subsequently adds subunits to the 39 end of the short RNA DNA primase periodically catalyzes the synthesis of a new
primer. Specific enzymes later degrade the primer (discussed RNA primer on the lagging strand as more of its template
in the next section), and the space fills in with DNA. strand becomes unwound by the helicase. A separate RNA
5′ 3′
3′ 3′
5′ 5′
A T A T
Base
3′ C G C G
5′
3′ G C G C
5′
Nucleotide 3′
joined to growing 5′ A T A
chain by DNA T OH
polymerase.
Phosphates
C C
released.
OH
3′
3′ 5′ 5′
Figure 12-14 A simplified view of DNA replication

One nucleotide at a time is added to the 39 end of a growing chain.
Leading strand template Replication fork 1 DNA primase enzymes initiate DNA
3′ replication by forming RNA primers on
both strands at a replication fork. Both
5′ 3′ strands require RNA primer for initiation
5′ of synthesis because DNA can be
RNA primers elongated only by addition to the 3′ end
5′ of the existing polynucleotide strand.
3′
DNA primase
3′
Direction of
DNA unwinding
5′
DNA polymerase Leading strand

Replication fork 2 DNA polymerase extends the leading
3′ and lagging strand copies from the RNA
primers in a 5′ 3′ direction.
5′ 3′
5′
RNA
RNA 5′
3′
3′ Direction of
DNA unwinding
5′ Lagging strand
Leading strand Replication fork 3 The leading strand is synthesized

3′ continuously in the direction toward the
replication fork; the lagging strand is
5′ 3′ synthesized in the direction away from
5′ the replication fork as short Okazaki
RNA Okazaki fragment fragments. Each Okazaki fragment
5′ synthesis begins with the synthesis of an
Lagging strand RNA 3′
RNA primer. Note that the first Okazaki
3′ Direction of fragment synthesized is at the far left
DNA unwinding and has become incorporated into the
5′ RNA lagging strand.
Leading strand Replication fork 4 After each Okazaki fragment has been
3′ elongated by DNA polymerase, the RNA
primer is degraded and gaps are filled in
5′ 3′ with DNA by a separate DNA polymerase,
5′ leaving a nick between the 5′ and 3′
RNA DNA polymerase, exonuclease
ends of the two adjoining fragments.
RNA 5′
Lagging strand 3′
3′ Direction of
DNA unwinding
5′
Leading strand Replication fork 5 The Okazaki fragments are linked

3′ together by a DNA ligase enzyme that
repairs the nick to form a continuous
5′ 3′ strand.
5′
RNA
Nick RNA 5′
Lagging strand 3′
3′ Direction of
DNA unwinding Figure 12-15 An overview of DNA
5′
replication
DNA ligase
primer initiates each Okazaki fragment, which DNA poly- joined by DNA ligase, an enzyme that links the 39 hydroxyl of
merase then extends toward the 59 end of the previously syn- one Okazaki fragment to the 59 phosphate of the DNA imme-
thesized fragment. When the RNA primer of the previously diately next to it, forming a phosphodiester linkage. (As you
synthesized fragment is reached, DNA polymerase degrades will see later in the chapter, DNA ligase also rejoins broken
and replaces the primer with DNA. The fragments are then phosphodiester bonds during DNA repair.)
266 / Chapter 12
DNA synthesis is bidirectional When double-stranded In bacteria each circular DNA molecule usually has only one
DNA separates, two replication forks form, creating a replica- origin of replication, so the two replication forks proceed
tion bubble. Within this bubble the molecule replicates in both around the circle and eventually meet at the other side to com-
directions from the origin of replication. FIGURE 12-16a shows plete the formation of two new DNA molecules.
how the lagging and leading strands are arranged at the two By contrast, a eukaryotic chromosome consists of one
replication forks. long, linear DNA molecule, so having multiple origins of rep-
Unlike the linear DNA molecules found in eukaryotic lication speeds the replication process (FIG. 12-16c and d). (In
cells, DNA in bacteria are in the form of circles, with no free mammals, between 20,000 and 50,000 origins of replication
ends. FIGURE 12-16b shows a replicating plasmid in bacteria. are involved in the replication of the DNA during a cell cycle.)
Plasmids are small, circular DNA molecules that carry genes Synthesis continues at each replication fork until it meets a
separate from those on a bacterial chromosome. Because plas- newly synthesized strand coming from the opposite direction.
mids are so small compared to the circular bacterial chromo- The result is a chromosome containing two DNA double heli-
some, they can be clearly photographed during replication. ces, each corresponding to a chromatid.
Replication bubble
Origin of replication
3′ 5′ 3′ 5′
3′ 5′
5′ Lagging strand Leading strand
3′ 3′
5′ Leading strand Lagging strand 5′ 3′
5′ 3′ 5′ 3′
Replication fork Replication fork
movement movement
(a) Leading and lagging strand synthesis at the two replication forks of a replication bubble.
Template DNA New DNA

(light blue) (dark blue)
3′
5′
5′
3′
(b) Most bacterial plasmids and chromosomes have only one origin of replication. Because DNA synthesis proceeds from that point in
both directions, two replication forks form (black arrows), travel around the circle, and eventually meet to form two chromosomes.
Kriegstein, H.J., and D.S. Hogness, “Mechanism of DNA
Replication in Drosophila Chromosomes: Structure of
Replication Forks and Evidence for Bidirectionality,”
Proceedings of the National Academy of Sciences
5′
3′ Single replication
Replication bubble formed from
bubbles two merged
U.S.A., Vol. 71 (1974): 135–139.
bubbles
3′
5′ Replication fork
340 nm
(c) TEM of two replication forks (black (d) Eukaryotic chromosome DNA contains multiple origins of replication.
arrows) in a segment of a eukaryotic DNA synthesis proceeds in both directions from each origin until adjacent
chromosome that has partly replicated. replication bubbles eventually merge.
Figure 12-16 Bidirectional DNA replication in bacteria and eukaryotes
Enzymes proofread and repair errors do not contain protein-coding genes. Telomeres consist of
in DNA short, non-coding, guanine-rich DNA sequences that repeat
many times (FIG. 12-18b). For example, in human sperm and
DNA replication occurs only once during each cell generation, egg cells, the sequence 59—TTAGGG—39 is repeated more
and it is important that the process be as accurate as possible than 1000 times at the ends of each chromosome. Therefore,
to avoid harmful, or possibly even lethal, mutations. Although although a small amount of telomeric DNA fails to replicate
base pairing during DNA replication is very accurate, errors each time the cell divides, a cell can divide many times before
do occur. Mechanisms have evolved that ensure that errors in it starts losing essential genetic information.
replication are corrected. During replication, DNA polymer- Telomerase, a special DNA replication enzyme, can
ases proofread each newly added nucleotide against its tem- lengthen telomeric DNA by adding back these repetitive
plate nucleotide. When an error in base pairing is found, DNA nucleotide sequences to the ends of eukaryotic chromosomes.
polymerase immediately removes the incorrect nucleotide This enzyme—which was discovered in 1984 by 2009 Nobel
and inserts the correct one. A few uncorrected mutations still laureates Elizabeth Blackburn and Carol Greider—is typi-
occur, but they are very infrequent, on the order of one error cally active in cells that divide an unlimited number of times,
for every 109 or 1010 base pairs. including protozoa and other unicellular eukaryotes, and most
When errors have been left uncorrected by the normal types of cancer cells. In humans and other mammals, active
repair activities of DNA polymerase during DNA replication, telomerase is usually present in germ line cells (which give rise
cells make use of other repair mechanisms (although exactly to eggs and sperm) and rapidly dividing cells (such as blood
how DNA repair enzymes identify these rare, often-subtle cells, cells lining the intestine, and skin cells), but not in most
errors in the vast amount of normal DNA is not well under- somatic cells of adult tissues.
stood). In mismatch repair special enzymes recognize the When most cells divide for repair or replacement, their
incorrectly paired nucleotides and remove them; DNA poly- chromosome ends shorten. Research evidence suggests that
merases then fill in the missing nucleotides. The observation the shortening of telomeres may contribute to cell aging and
that individuals with a hereditary defect in a mismatch repair apoptosis, which is programmed cell death. The pioneering
enzyme are likely to develop a type of colon cancer demon- studies of U.S. biologist Leonard Hayflick in the 1960s showed
strates the crucial role of mismatch repair enzymes in ensuring that when normal somatic cells of the human body are grown
the fidelity of DNA replication from one generation to the next. in culture, they lose their ability to divide after a limited num-
In Chapter 13 you will discover that some types of radia- ber of cell divisions. Furthermore, the number of cell divi-
tion and chemicals, both in the environment and within sions is determined by the age of the individual from whom
the cells themselves, cause mutations in DNA. These muta- the cells were taken. Cells from a 70-year-old can divide only
tions are almost always harmful, and cells usually correct
mutations by using one or more DNA repair enzymes. About Nuclease enzyme
bound to DNA DNA lesion
100 kinds of repair enzymes in the bacterium E. coli, and 130
kinds in human cells, have been discovered so far.
One type of DNA repair—nucleotide excision repair—is 5′ 3′
commonly used to repair DNA lesions (deformed DNA) caused 3′ 5′
by the sun’s ultraviolet radiation or by harmful chemicals
(FIG. 12-17). Three enzymes are involved in nucleotide excision
repair: a nuclease to cut out the damaged DNA, a DNA poly-
1 Mispaired or altered bases are
merase to add the correct nucleotides, and DNA ligase to close detected by groups of repair
the breaks in the sugar–phosphate backbone. Individuals suf- enzymes that scan the DNA for
fering from the disease xeroderma pigmentosum have an inher- lesions. A nuclease cuts out a
ited defect in a nucleotide excision repair enzyme. Affected piece of DNA, including the
damaged part.
individuals develop many skin cancers at an early age because
DNA lesions caused by ultraviolet radiation are not repaired.
5′ 3′
Telomeres cap eukaryotic chromosome 3′ 5′
ends DNA polymerase

2 DNA polymerase and DNA
DNA ligase ligase complete the repair by
Unlike bacterial DNA, which is circular, eukaryotic chromo- synthesizing the removed part
somes have free ends. Because DNA replication is discontinu- and sealing the new DNA
ous in the lagging strand, DNA polymerases do not complete New DNA
to the original DNA.
replication of the strand neatly. At the end of the DNA, a 5′ 3′
small portion is left unreplicated, and a small, single-strand 3′ 5′
segment of the DNA is lost with each cell cycle (FIG. 12-18a).
The important genetic information is retained because Figure 12-17 Nucleotide excision repair of damaged DNA
chromosomes have protective end caps, or telomeres, that
268 / Chapter 12
Unwound DNA before replication
5′ 3′
3′ 5′
1 When DNA replication occurs, the

5′ end of each newly synthesized
strand contains a short segment
of RNA. This RNA functions as a
primer on the lagging strand.
5′ 3′
3′ 5′
RNA primer + RNA primer
5′ 3′
Peter Lansdorp/Visuals Unlimited, Inc.

3′ 5′
2 There is no way to prime the last

section of the exposed template
strand after the last RNA primer is
removed. The result is the loss of a
short, non-coding telomere sequence
with each cell cycle, and it produces a
DNA molecule with a shorter 5′ end 10 mm
than in the original chromosome.
5′ 3′ (b) Repetitive telomere sequences cap the ends of linear DNA in
eukaryotic chromosomes. LM of duplicated human chromosomes
3′ 5′ with fluorescent-tagged telomeres (yellow).
+
5′ 3′ cell division also has potentially serious consequences. For
example, if transplanted into the body, cells with active telom-
3′ 5′
erase might behave like cancer cells.
(a) Eukaryotic DNA replication shortens the ends of chromosomes
Cancer cells have the ability to divide hundreds of times in
each generation. culture; in fact, they are virtually immortal. Researchers have
demonstrated that in cancer cells of the prostate and pancreas,
Figure 12-18 Telomeres telomere DNA sequences shorten to a critical point at which
Repetitive telomere sequences protect the ends of eukaryotic time telomerase is reactivated, which may explain their ability
chromosomes as they shorten during DNA replication to proliferate in a rapid and uncontrolled manner. Most cancer
cells, including human cancers of the breast, lung, colon, pros-
tate gland, and pancreas, have active telomerase, which allows
20 to 30 times, compared with those from an infant, which them to maintain telomere length and possibly to resist apop-
can divide 80 to 90 times. tosis. Given that most adult differentiated cells have very low
Many investigators have observed correlations between telomerase activity, there are now many types of cancer drugs in
telomerase activity and the ability of cells to undergo unlim- development that target the telomerase enzyme as an anticancer
ited divisions without showing signs of the aging process. therapy.
However, the evidence of a causal relationship was not found
until the early 2000s, when scientists used recombinant DNA
technology (see Chapter 15) to infect cultured normal human
cells with a virus that carried the genetic information coding
for the catalytic subunit of telomerase. The infected cells not checkpoint 12.3
only produced active telomerase, which elongated the telo- • connect How did the ability to distinguish old and
meres significantly, but also continued to divide long past the newly synthesized DNA strands enable Meselson and Stahl
point at which cell divisions would normally cease. to verify that DNA replication is semiconservative?
Telomeres and telomerase are an active focus of research, • What feature of DNA structure causes DNA replication to
for both scientific and practical reasons. The ability to give be continuous for one strand but discontinuous for the
somatic cells telomerase so that they can divide many times other?
more than they ordinarily would has many potential thera- • connect What is the reason that eukaryotic cells
peutic applications, especially if lost or injured cells must be require telomerase, but bacterial cells do not?
replaced. However, giving such cells the property of unlimited
Summary: Focus on Learning Objectives
12.1 Evidence of DNA as the Hereditary Material two hydrogen bonds with thymine (T), and guanine
(page 254) (G) forms three hydrogen bonds with cytosine (C).
1 Summarize the evidence that accumulated during the 1940s and • Complementary base pairing between A and T and
early 1950s demonstrating that DNA is the genetic material. between G and C is the basis of Chargaff’s rules, which
• Many early geneticists thought genes were made of prostate that A equals T and that G equals C.
teins. They knew proteins were complex and variable, • Because complementary base pairing holds together the
whereas they thought nucleic acids were simple molecules two strands of DNA, it is possible to predict the base
with a limited ability to store information. sequence of one strand if you know the base sequence of
• Several lines of evidence supported the idea that DNA the other strand.
(deoxyribonucleic acid) is the genetic material. In 12.3 DNA Replication (page 261)
transformation experiments, the DNA of one strain of bacte- 6 Cite evidence from Meselson and Stahl’s experiment that
ria can endow related bacteria with new genetic characteristics. enabled scientists to differentiate between semiconservative
• When a bacterial cell becomes infected with a replication of DNA and alternative models.
bacteriophage (virus), only the DNA from the virus enters • When E. coli cells are grown for many generations in a
the cell; this DNA is sufficient for the virus to reproduce medium containing heavy nitrogen ( 15 N), they incorporate
and form new viral particles. the 15 N into their DNA. When researchers transfer cells from
2 State the questions that these classic experiments addressed: a 15 N medium to a 14 N medium and isolate them after either
Griffith’s transformation experiment, Avery’s contribution to one or two generations, the density of the DNA in each group
Griffith’s work, and the Hershey–Chase experiments. is what would be expected if DNA replication were semicon-
• Griffith’s transformation experiment addressed this ques- servative. In semiconservative replication each daughter
tion: Can a genetic trait be transmitted from one bacterial double helix consists of an original strand from the parent
strain to another? (The answer is yes.) molecule and a newly synthesized complementary strand.
• Avery’s experiments addressed this question: What molecule is 7 Summarize how DNA replicates and identify some unique
responsible for bacterial transformation? (The answer is DNA.) features of the process.
• The Hershey–Chase experiments addressed this question: • During DNA replication, the two strands of the double
Is DNA or protein the genetic material in bacterial viruses helix unwind. Each strand serves as a template for form-
(phages)? (The answer is DNA.) ing a new, complementary strand. Replication is initiated
as DNA primase synthesizes a short RNA primer. DNA
12.2 The Structure of DNA (page 257) polymerase then adds new nucleotide subunits to the
3 Explain how nucleotide subunits link to form a single DNA strand. growing DNA strand.
• Watson and Crick’s model of the structure of DNA dem- • Additional enzymes and other proteins are required to
onstrated how information can be stored in the molecule’s unwind and stabilize the separated DNA helix. DNA
structure and how DNA molecules can serve as templates helicases open the double helix, and topoisomerases
for their own replication. prevent tangling and knotting.
• DNA is a polymer of nucleotides. Each nucleotide subunit 8 Explain the complexities of DNA replication that make the
contains a nitrogenous base, which may be one of the process (a) bidirectional and (b) continuous in one strand and
purines (adenine or guanine) or one of the pyrimidines discontinuous in the other.
(thymine or cytosine). Each base covalently links to a • DNA replication is bidirectional, starting at the origin of
pentose sugar, deoxyribose, which covalently bonds to a replication and proceeding in both directions from that
phosphate group. point. A eukaryotic chromosome may have multiple origins
• The backbone of each single DNA chain is formed by alter- of replication and may be replicating at many points along
nating sugar and phosphate groups, joined by covalent its length at any one time.
phosphodiester linkages. Each phosphate group attaches • DNA synthesis always proceeds in a 5′ → 3′ direction,
to the 59 carbon of one deoxyribose and to the 39 carbon which requires that one DNA strand, the lagging
of the neighboring deoxyribose. strand, be synthesized discontinuously, as short Okazaki
4 Describe how the two strands of DNA are oriented with fragments. DNA primase synthesizes short RNA primers
respect to each other. on the lagging strand, and DNA ligase links Okazaki frag-
• Each DNA molecule consists of two polynucleotide chains that ments of newly synthesized DNA. The opposite strand, the
associate as a double helix. The two chains are antiparallel leading strand, is synthesized continuously.
(running in opposite directions); at each end of the DNA 9 Discuss how enzymes proofread and repair errors in DNA.
molecule, one chain has a phosphate attached to a 59 deoxy- • During replication, DNA polymerases proofread each newly
ribose carbon, the 59 end, and the other has a hydroxyl group added nucleotide against its template nucleotide. When an
attached to a 39 deoxyribose carbon, the 39 end. error in base pairing is found, DNA polymerase immediately
5 State the base-pairing rules for DNA and describe how removes the incorrect nucleotide and inserts the correct one.
complementary bases bind to each other. • In mismatch repair enzymes recognize incorrectly paired
• Hydrogen bonding between specific base pairs holds nucleotides and remove them; DNA polymerases then fill in
together the two chains of the helix. Adenine (A) forms the missing nucleotides.
270 / Chapter 12
• Nucleotide excision repair is commonly used to repair slightly with each cell cycle but can be extended by the
DNA lesions caused by the sun’s ultraviolet radiation or by enzyme telomerase.
harmful chemicals. Three enzymes are involved: a nuclease • The absence of telomerase activity in certain cells may be a
to cut out the damaged DNA, a DNA polymerase to add cause of cell aging, in which cells lose their ability to divide
the correct nucleotides, and DNA ligase to close the breaks after a limited number of cell divisions.
in the sugar–phosphate backbone.
• Most cancer cells, including human cancers of the
10 Define telomere and describe the possible connections between breast, lung, colon, prostate gland, and pancreas, have
telomerase and cell aging and between telomerase and cancer. active telomerases to maintain telomere length and
• Eukaryotic chromosome ends, called telomeres, are short, possibly to resist apoptosis, which would normally occur
non-coding, repetitive DNA sequences. Telomeres shorten in an aging cell.
Test Your Understanding

Know and Comprehend base sequences (d) some portions of a single DNA strand are
1. When Griffith injected mice with a combination of live old and other portions are newly synthesized (e) each double
rough-strain and heat-killed smooth-strain pneumococci, he helix consists of one old and one newly synthesized strand
discovered that (a) the mice were unharmed (b) the dead 9. Topoisomerases (a) synthesize DNA (b) synthesize RNA
mice contained living rough-strain bacteria (c) the dead mice primers (c) join Okazaki fragments (d) break and rejoin DNA
contained living smooth-strain bacteria (d) DNA had been to reduce torsional strain (e) prevent single DNA strands
transferred from the smooth-strain bacteria to the mice from joining to form a double helix
(e) DNA had been transferred from the rough-strain bacteria 10. A lagging strand forms by (a) joining primers (b) joining
to the smooth-strain bacteria Okazaki fragments (c) joining leading strands (d) breaking up
2. Which of the following inspired Avery and his colleagues to a leading strand (e) joining primers, Okazaki fragments, and
perform the experiments demonstrating that the transform- leading strands
ing factor in bacteria is DNA? (a) that A is equal to T and 11. The immediate source of energy for DNA replication is
that G is equal to C (b) Watson and Crick’s model of DNA (a) the hydrolysis of nucleotides with three phosphate
structure (c) Meselson and Stahl’s studies on DNA replica- groups (b) the oxidation of NADPH (c) the hydrolysis of ATP
tion in E. coli (d) Griffith’s experiments on smooth and rough (d) electron transport (e) the breaking of hydrogen bonds
strains of pneumococci (e) Hershey and Chase’s experiments 12. Which of the following statements about eukaryotic
on the reproduction of bacteriophages chromosomes is false? (a) eukaryotic chromosomes have
3. In the Hershey–Chase experiment with bacteriophages, free ends (b) telomeres contain protein-coding genes
(a) harmless bacterial cells permanently transformed into vir- (c) telomerase lengthens telomeric DNA (d) telomere
ulent cells (b) DNA was shown to be the transforming factor shortening may contribute to cell aging (e) cells with active
of earlier bacterial transformation experiments (c) the replica- telomerase may undergo many cell divisions
tion of DNA was conclusively shown to be semiconservative
(d) viral DNA was shown to enter bacterial cells and cause Apply and Analyze
the production of new viruses within the bacteria (e) viruses 13. Many cancer cells are immortal and can be cultured in the
injected their proteins, not their DNA, into bacterial cells laboratory for many years. Many of these cell lines have
4. The two complementary strands of the DNA double helix highly active telomerase activities. Why would that affect the
are held to each other by (a) ionic bonds between deoxyri- ability of cancer cells to sustain growth and cell division?
bose molecules (b) ionic bonds between phosphate groups 14. visualize Construct a diagram of a replication fork.
(c) covalent bonds between nucleotide bases (d) covalent Label the 39 and 59 ends of the leading strand, lagging
bonds between deoxyribose molecules (e) hydrogen bonds strand, and the two strands of the DNA double helix.
between nucleotide bases
5. If a segment of DNA is 5′ — CATTAC — 3′ , the Evaluate and Synthesize
complementary DNA strand is (a) 3′ — CATTAC — 5′ 15. Explain to a friend the characteristics a molecule must have
(b) 3′ — GTAATG — 5′ (c) 5′ — CATTAC — 3′ to function as genetic material and then explain the impor-
(d) 5′ — GTAATG — 3′ (e) 5′ — CATTAC — 5′ tant features of the structure of DNA that are consistent
6. Each DNA strand has a backbone that consists of alternating with its role as the chemical basis of heredity.
(a) purines and pyrimidines (b) nucleotide bases (c) hydrogen 16. interpret data In the Hershey–Chase experiment,
bonds and phosphodiester linkages (d) deoxyribose and the radioactive label 32P was present inside bacterial cells
phosphate (e) phosphate and phosphodiester linkages (i.e., in the pellet), whereas the radioactive label 35 S was
7. The experiments in which Meselson and Stahl grew bacteria present outside bacterial cells (in the supernatant). What
in heavy nitrogen conclusively demonstrated that DNA (a) is would the researchers have concluded had the reverse been
a double helix (b) replicates semiconservatively (c) consists of true, that is, if the radioactive label 35 S were inside the cells
repeating nucleotide subunits (d) has complementary base and the radioactive label 32P were outside the cells?
pairing (e) is always synthesized in the 5′ → 3′ direction 17. evolution link How does DNA being the universal
8. The statement “DNA replicates by a semiconservative molecule of inheritance in all cells support evolutionary theory?
mechanism” means that (a) only one DNA strand is copied
(b) first one DNA strand is copied and then the other strand For access to MindTap and additional study materials visit
is copied (c) the two strands of a double helix have identical www.cengagebrain.com.

DNA The Carrier of Genetic Information SL and HL

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12 DNA: The Carrier of Genetic Information

After the rediscovery of Mendel’s principles in 1900, geneticists con-

hypothesis : The ability of pneumococ- Experiment 1 Experiment 2 Experiment 3 Experiment 4

methionine and that nucleic acids contain

that the protein had not entered the cells.

DNA: The Carrier of Genetic Information / 255

hypothesis : DNA is the genetic material in bacterial viruses.

Experiment A, phages Bacteria in No radioactively labeled

Bacteria in pellet Some newly assembled

Figure 12-3 The Hershey–Chase experiments

DNA: The Carrier of Genetic Information / 257

DNA is made of two polynucleotide

National Library of Medicine/Science Source

why is it used? X-ray diffraction can be used to determine the

A. Barrington Brown/Science Source

X-ray source DNA sample

2. X-ray diffraction image of DNA. This diagonal pattern of spots

(c) Hypothesis 3: Dispersive replication

Figure 12-11 The Meselson–Stahl experiment A T A T A T A T

Figure 12-12 The perpetuation of a mutation

Single-strand Origin of replication

Figure 12-13 DNA strand separation during DNA replication

Figure 12-14 A simplified view of DNA replication

DNA: The Carrier of Genetic Information / 265

DNA polymerase Leading strand

Leading strand Replication fork 3 The leading strand is synthesized

Leading strand Replication fork 5 The Okazaki fragments are linked

Template DNA New DNA

Figure 12-16 Bidirectional DNA replication in bacteria and eukaryotes

DNA: The Carrier of Genetic Information / 267

ends DNA polymerase

1 When DNA replication occurs, the

Peter Lansdorp/Visuals Unlimited, Inc.

2 There is no way to prime the last

Test Your Understanding

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2. X-ray diffraction image of DNA. This diagonal pattern of spots