14

DNA STRUCTURE, REPLICATION, AND ORGANIZATION

Chapter Outline
WHY IT MATTERS
14.1 ESTABLISHING DNA AS THE HEREDITARY MOLECULE
Griffith found a substance that could transform pneumonia bacteria genetically.
Avery and his coworkers identified DNA as the molecule that transforms nonvirulent Streptococcus bacteria to
the virulent form.
Hershey and Chase obtained the final experimental evidence establishing DNA as the hereditary molecule.
14.2 DNA STRUCTURE
Watson and Crick brought together information from several sources to develop a model for DNA structure.
The new model proposed that two polynucleotide chains wind into a DNA double helix.
14.3 DNA REPLICATION
The Meselson and Stahl experiment showed that DNA replication is semiconservative.
DNA polymerases are the primary enzymes of DNA replication.
Helicases unwind DNA for new DNA synthesis, and other proteins stabilize the DNA at the replication fork.
RNA primers provide the starting point for DNA polymerase to begin synthesizing a new DNA chain.
One new DNA strand is synthesized continuously; the other, discontinuously.
Multiple enzymes coordinate their activities in DNA replication.
Key proteins and enzymes for replication are assembled into a complex called a replisome.
Bacterial chromosomes have a single replcation origin; eukaryotic chromosomes have multiple replication
origins.
Telomerases solve a specialized replication problem at the ends of linear DNA molecules in eukaryotes.
In eukaryotes, newly replicated DNA is assembled into nucleosomes.
MOLECULAR INSIGHTS: DNA REPLICATION IN HUMANS: LOADING AND UNLOADING THE SLIDING
CLAMP.
THINK OUTSIDE THE BOOK
14.4 REPAIRS OF ERRORS IN DNA
Proofreading depends on the ability of DNA polymerases to reverse and remove mismatched bases.
A mismatch repair mechanism corrects replication errors that escape proofreading.
Excision repair mechanisms correct various kinds of DNA damage.
Replication errors and DNA damage that remain unrepaired are mutations.
UNANSWERED QUESTIONS

Learning Objectives
14.1 Describe the experiments that led to the establishment of DNA as the genetic material.
14.1.1 Diagram Griffith's experiments with pneumococcus and his discovery of the transforming principle.
14.1.2 Diagram Avery's experiments with Streptococcus that identified the transforming principle.
14.1.3 Diagram Hershey and Chase's experiments that identified DNA as the hereditary molecule.

14.2 Review the double-helix model of DNA proposed by Watson and Crick.
14.2.1 Discuss the experimental evidence that led to the discovery of the double- helix model of DNA.

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 14.2.2 Illustrate the structural features of the DNA double helix.

14.3 Summarize the steps involved in DNA replication.

14.3.1 Compare the three theoretical models of DNA replication.
14.3.2 Illustrate the method used by Meselson and Stahl to show that DNA replication is semiconservative.
14.3.3 Outline the roles of DNA polymerases and the other major enzymes in DNA replication.
14.3.4 Specify the four key molecular events that occur during DNA replication.
14.3.5 Illustrate the synthesis of leading and lagging strands in the two replication forks of a replication bubble.
14.3.6 Explain the significance of the replisome.
14.3.7 Compare eukaryotic and prokaryotic replication origins.
14.3.8 Explain the significance of telomeres and the role of telomerases.
14.3.9 Examine the structure and significance of eukaryotic nucleosomes.

14.4 Review DNA base-pair mismatches and the three types of repair mechanisms used to correct them.
14.4.1 Demonstrate the three basic steps common to DNA repair mechanisms.
14.4.2 Outline the role of DNA polymerase in proofreading.
14.4.3 Illustrate how the mismatch repair mechanism corrects post-replication errors.
14.4.4 Diagram how base- and nucleotide-excision repair mechanisms correct errors caused by DNA damage.
14.4.5 Explain the consequences of unrepaired replication errors and DNA damage.

Key Terms
deoxyribonucleic acid X-ray diffraction primer replication bubble
(DNA) double-helix model primase telomeres
bacteriophages complementary base discontinuous telomerase
(phages) pairing replication base-pair mismatch
transformation antiparallel Okazaki fragments proofreading
adenine semiconservative leading strand mismatch repair
guanine replication leading strand template excision repair
thymine sliding DNA clamp lagging strand base-excision repair
cytosine origin of replication lagging strand template nucleotide-excision
Chargaff’s rule DNA helicase DNA polymerase III repair
sugar-phosphate replication fork DNA polymerase I mutations
backbone single-stranded binding DNA ligase
5′ end proteins
replisome
3′ end topoisomerase

Lecture Outline
Why It Matters
A. Swiss physician and chemist Johann Miescher was the first to study the chemical composition of the cell
nucleus.
1. In the spring of 1868, Miescher used pus from discarded bandages to collect white blood cells.
2. He discovered an acidic substance high in phosphorus and called it nuclein.
B. DNA is the genetic material of all living organisms.
1. DNA was recognized as the hereditary molecule in 1952.
2. DNA structure was discovered by Watson and Crick (Figure 14.1) and was key to understanding how
DNA is used in a cell.

14.1 Establishing DNA as the Hereditary Molecule

1. In the first half of the 20th century, many scientists believed proteins were the most likely candidates for
hereditary molecules; some believed nucleic acids were the hereditary molecules.
B. Griffith found a substance that could transform pneumonia bacteria genetically.
1. Griffith could use “components” from dead bacteria to transform live bacteria.
a. S strains of virulent bacteria were heat killed by fire.
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 b. R strains of nonvirulent bacteria were converted to virulent bacteria when exposed to leftover
“components” of dead S strains.
2. Transformed bacteria retained characteristics and passed them on to their offspring.
a. Griffith concluded that some molecule was responsible for the transformation.
b. Griffith concluded that the molecule could be passed to future generations.
C. Avery and his coworkers identified DNA as the molecule that transforms nonvirulent Streptococcus
bacteria to the virulent form.
1. Avery and his coworkers, MacLeod and McCarty, in 1944 repeated Griffith’s experiments, using
isolated components from the dead bacteria and enzymes to degrade those components (i.e., protein
and DNA degrading enzymes).
2. Avery could inhibit transformation when DNA-degrading enzymes were used and concluded that DNA
was the transforming material.
D. Hershey and Chase found the final experimental evidence establishing DNA as the hereditary molecule
(Figure 14.3).
1. They used bacteriophages, or viruses that infect bacteria, to demonstrate this.
2. Hershey and Chase radiolabeled DNA and protein components of bacteriophages and examined
infected cells.
3. Only radiolabeled DNA is observed in infected cells, thus proving that DNA is the hereditary
molecule.

14.2 DNA Structure

1. Following the above experiments, a highly competitive scientific race to discover the structure of DNA
began.
A. Watson and Crick brought together information from several sources to develop a model for DNA
structure.
1. Previous research deciphered the building blocks of DNA.
a. DNA is made of four possible types of nucleotides.
b. Each nucleotide is made of a five-carbon sugar, a phosphate group, and one of four
nitrogenous bases (adenine, thymine, guanine, or cytosine).
(1) Adenine and guanine are purine nucleotides.
(2) Thymine and cytosine are pyrimidine nucleotides.
2. Chargaff discovered that purine and pyrimidine bases occur in equal ratios.
a. Adenine equals thymine.
b. Guanine equals cytosine.
3. Other research determined DNA contains nucleotides joined to form polynucleotide chains (Figure
14.4).
a. Nucleotides are oriented such that the 5' OH group on one is attached to the 3' OH group on
another, with the phosphate group in between (i.e., sugar–phosphate–sugar).
b. Nucleotides are held together by strong phosphodiester bonds.
4. Wilkins and Franklin used X-ray diffraction techniques to examine the arrangement of molecules in
DNA (Figure 14.5).
a. Patterns indicated that DNA molecules were about 2 nm in diameter.
b. Patterns also indicated that DNA is helical in shape.
B. The new model proposed that two polynucleotide chains wind into a DNA double helix (Figure 14.6).
1. Their model proposed DNA was actually two strands connected by the nitrogen base component of
opposing nucleotides (hydrogen bonds).
a. Based on size constraints, A–T and G–C pairs on opposing strands fit the model of likely
complementary pairing (as suggested by Chargaff’s data).
b. Similarly, the two strands of nucleotides fit properly when oriented antiparallel to one another
(i.e., 3' end of one strand opposite the 5' end of the other strand).
2. The two strands twist around one another to form a right-handed double helix.
a. The strands are held by hydrogen bonds.
b. Each base pair occupies 0.34 nm of space in the length of the strands, and 10 base pairs
occupy the length before a turn of the helix (every 3.4 nm).
3. Watson and Crick realized the blueprint held within DNA.
a. The only difference in DNA strands are the nitrogenous bases (A, T, G, C) of the nucleotides.

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 b. Watson and Crick proposed that the sequence of nucleotides in a DNA strand might be the
“code” for our traits.

14.3 DNA Replication

1. Data on DNA structure was used to suggest how DNA replicates.
a. Base pair differences (sequences) appeared key to the “code” in DNA.
2. Watson and Crick suggested that the key to replication had to do with repeating the nucleotides while
somehow retaining the original sequence.
a. Suggested strands must somehow unwind to reveal the sequence of nucleotides.
b. Each strand serves as a “template” for replication.
3. Semiconservative replication is suggested as a method of DNA replication (Figure 14.7).
a. Watson and Crick suggested that each DNA strand is maintained and used as a template to make
two new strands of DNA.
i. In this method, an old DNA strand then becomes paired with a newly made DNA
strand.
ii. In other suggested methods, DNA strands may have all old or new DNA strands
combining (conservative replication) or a mosaic of old and new strands (dispersive replication)
(Figure 14.8).
A. The Meselson and Stahl experiment showed that DNA replication is semiconservative (Figure 14.9).
1. A “heavy” DNA double helix was made using nucleotides containing all N 15 molecules.
a. The heavy DNA was then allowed to replicate in medium containing only nucleotides
containing “light” (i.e., N14) nucleotides.
b. Centrifugation of the resulting DNA strands demonstrated that an original strand ends up
attached to a new strand following replication.
B. DNA polymerases are the primary enzymes of DNA replication.
1. DNA polymerases use nucleoside triphosphates (dATP, dGTP, dCTP, or dTTP) as substrates for
making new DNA strands.
2. The polymerase recognizes the nucleotide on the template strand and positions the proper
complementary nucleoside triphosphate in the growing new strand.
3. The polymerase can only add a nucleotide triphosphate to the 3' OH end of an existing nucleotide
strand (Figure 14.10).
4. The key molecular events of DNA replication are:
a. Two strand of DNA molecule unwind for replication to occur.
b. Nucleotides are added only to an existing chain.
c. The overall direction is from the 5' to the 3' direction.
d. Nucleotides add according to the A–T and G–C base pairing rules.
C. Helicases unwind DNA for new DNA synthesis, and other proteins stabilize the DNA at the replication
fork. (See Table 14.1 for a list of major enzymes of replication.)
1. DNA helicase helps unwind the DNA strands exposing the nucleotide bases (Figure 14.12).
2. Single-stranded binding proteins stabilize the open DNA strands for the replication process.
3. Topoisomerases help undo any twisting that occurs in the DNA strands as they are unwound.
D. RNA primers provide the starting point for DNA polymerase to begin synthesizing a new DNA chain.
1. A primase enzyme adds the first nucleotide (primer) to the new strand.
a. The primer is a short strand of RNA.
b. The primer is later removed and replaced by DNA before replication is complete.
E. One new DNA strand is synthesized continuously; the other discontinuously.
1. DNA polymerases can catalyze the synthesis of one strand continuously (i.e., in the 5' to 3' direction,
which is considered the “leading” strand).
2. Because of its orientation, the other strand is replicated in pieces or discontinuously (Figure 14.14).
a. The polymerase must detach and reattach as it replicates this strand (lagging strand).
b. The short DNA fragments added are referred to as Okazaki fragments.
F. Multiple enzymes coordinate their activities in DNA replication (Figure 14.15).
1. The helicase unwinds the DNA strands first.
2. The primases then lay down the first primers at a site called the replication fork.
3. DNA polymerase then starts laying down other nucleotides.
4. If the polymerase reaches a site where a primer is, the polymerase leaves, and another special
polymerase binds the growing strand to remove the RNA primer.
5. DNA ligase helps close any nicks in the DNA strands once the primers are removed.
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 G. Key proteins and enzymes for replication are assembled into a complex called a replisome.
1. DNA replication begins at replication origins.
2. Replication can occur at multiple sites (or forks) on a chromosome.
3. Unwinding at an ori on a DNA molecule actually produces two replication forks (Figure 14.16).
4. The two forks join together to form a replication bubble.
5. In bacteria, there is one origin and the replication proceeds in two directions.
6. Even though eukaryotes have many long DNA molecules, sometimes the replication process can be
faster because there are hundreds of origins of replications on a single chromosome.
7. A replication bubble is formed around each origin.
H. Bacterial chromosomes have a single replication origin; eukaryotic chromosomes have multiple replication
origins.
1. Unwinding at an ori within a DNA molecule actually produces two replication forks.
2. For small circular genomes, such as those found in E. coli, and in many bacteria and archaea, there
is a single ori.
3. Eukaryotic genomes are distributed among several linear chromosomes.
I. Telomerases solve a specialized replication problem at the ends of linear DNA molecules in eukaryotes.
1. When starting replication at the end of a linear piece of DNA, removal of the primer results in a gap,
which normal DNA polymerases are unable to fix (Figure 14.18).
a. Over time, linear pieces of DNA will get shorter following each replication.
b. Eukaryotes have a special polymerase (telomerase) that adds nucleotides to the end of DNA
before replication.
2. Then, ends of eukaryotic DNA are referred to as telomeres since they usually have repeated DNA that
does not typically code for proteins.
a. Eukaryotic cells typically have a finite number of mitotic divisions they can undergo before
they stop dividing and die.
b. Telomerase activity may be linked to aging (underactive enzymes) and cancer (overactive
enzymes).
J. In eukaryotes, newly replicated DNA is assembled into nucleosomes.
1. The most fundamental structure of the eukaryotic chromosome is the nucleosome in which DNA wraps
around an eight-protein histone complex called the nucleosome core particle.
2. For replication to take place on a segment of DNA, first, nucleosomes disassemble from the
chromosome as the replication fork passes.
Molecular Insights: DNA replication in humans: Loading and unloading the sliding clamp.

Think Outside The Book

A. What are primers and why are they needed?.
B. What are the roles of DNA polymerase III and DNA polymerase I in DNA replication.
C. Why are telomeres important?

14.4 Repair of Errors in DNA

1. Most of the mistakes that occur, called base-pair mismatches, are corrected.
A. Proofreading depends on the ability of DNA polymerases to reverse and remove mismatched bases (Figure
14.21).
1. Kornberg and Brutlag described the ability of polymerases to work only if bases are paired correctly.
2. Error rates due to DNA polymerase mismatching is low (1 in 106).
B. A mismatch repair mechanism corrects replication errors that escape proofreading (Figure 14.22).
1. Mismatch repair systems search DNA for distortions that may be associated with incorrect base
pairings.
2. The mismatch is cut out and repaired with a DNA polymerase and a DNA ligase.
a. Faulty mismatch systems are associated with human disorders (e.g., Xeroderma
pigmentosum).
b. Differences that do not get fixed and persist in DNA are referred to as mutations.
C. Excision repair mechanisms correct various kinds of DNA damage.
1. Repair of nonbulky damage to bases occurs by a base-excision repair mechanism.
2. Some DNA damage is more extensive, causing bulky distortions in the DNA.
3. Nucleotide-excision repair is a mechanism that can repair DNA damage such as thymine dimmers.

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 a. The autosomal recessive disease in humans, xeroderma pigmentosum (XP), is caused by a genetic
defect in nucleotidee xcision repair.
D. Replication errors and DNA damage that remain unrepaired are mutations.
1. Few replication errors are uncorrected.
2. Errors that remain uncorrected are mutations.
a. mutations are highly important to the evolutionary process.

Unanswered Questions
A. Research is ongoing to understand basic cellular processes and to apply it to areas such as understanding
and treating human disease.
B. In humans, telomerase is active in cells, destined to form gametes and also in stem cells.
C. Telomerase is also active in most cancers, and these cells maintain a shorter telomere length as compared to
normal somatic cells.
D. How do cells measure the length of the telomeric DNA tract?
1. Several proteins have been identified that bind specifically to the telomeric repeat sequences and are
thought to regulate telomere length.
2. Shortened telomeres, whether due to cell division in the absence of telomerase or experimental
manipulations of telomere proteins, resemble broken chromosomes and can prevent the cell from
progressing through the cell cycle.
E. Are short telomeres involved in organismal aging?
F. Current research is examining the role of telomerase and telomeres to develop anticancer strategies.

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accessible website, in whole or in part.