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Chapter 6
DNA Structure,
Replication, and
Manipulation
1
Genome Size
• The genetic complement of a cell or virus constitutes
its genome.
• In eukaryotes, this term is commonly used to refer to
one complete haploid set of chromosomes, such as that
found in a sperm or egg.
• The C-value is the DNA content of the haploid
genome
• The units of length of nucleic acids in which genome
sizes are expressed :
• kilobase (kb) 103 base pairs
• megabase (Mb) 106 base pairs
Genome Size
• Viral genomes are typically in the range 100–1000 kb:
– Bacteriophage MS2, one of the smallest viruses, has
only four genes in a single stranded RNA molecule of
about 4000 nucleotides (4kb)
• Bacterial genomes are larger, in the range 1–10 Mb:
– The chromosome of Escherichia coli is a circular
DNA molecule of 4600 kb.
Genome Size
• Eukaryotic genomes are typically in the range 100–1000
Mb:
– The genome of a fruit fly, Drosophila melanogaster
is 180 Mb
• Among eukaryotes, genome size often differs
tremendously, even among closely related species
Table T01: Genome Size of Some Representative Viral, Bacterial, and
Eukaryotic Genomes
The C-value Paradox
• Genome size among species of protozoa differ by
5800-fold, among arthropods by 250-fold, fish 350fold, algae 5000-fold, and angiosperms 1000-fold.
• The C-value paradox: Among eukaryotes, there is no
consistent relationship between the C-value and the
metabolic, developmental, or behavioral complexity
of the organism
• The reason for the discrepancy is that in higher
organisms, much of the DNA has functions other
than coding for the amino acid sequence of proteins
Figure 06.F01_LEFT: An example of the C-value paradox. The Japanese pufferfish
Takifugu rubripes has a genome size of 400 Mb.
Left © Ken Lucas/Visuals Unlimited
Figure 06.F01_RGHT: An example of the C-value paradox. The two-toed salamander
Amphiuma means one of 90,000 Mb.
Right © Phil Dotson/Photo Researchers, Inc.
DNA: Chemical Composition
• DNA is a linear polymer of four deoxyribonucleotides
• Nucleotides composed of 2'- deoxyribose (a five-carbon
sugar), phosphoric acid, and the four nitrogen-containing
bases denoted A, T, G, and C
Figure 06.03: A typical nucleotide showing the three major components.
DNA: Chemical Composition
• Two of the bases, A and G, have a double-ring
structure; these are called purines
• The other two bases,T and C, have a single-ring
structure; these are called pyrimidines
Figure 06.02: Chemical structures of adenine, thymine, guanine, and cytosine.
DNA Structure
• The nucleotides are joined to form a polynucleotide
chain, in which the phosphate attached to the 5'
carbon of one sugar is linked to the hydroxyl group
attached to the 3' carbon of the next sugar in line
• The chemical bonds by which the sugar components
of adjacent nucleotides are linked through the
phosphate groups are called phosphodiester bonds
Figure 06.F03: A typical nucleotide showing the three major components, the
difference between DNA and RNA, and the distinction between nucleoside and
nucleotide.
Figure 06.04: Three nucleotides at the 5’-end of a single polynucleotide strand.
DNA Structure
• The duplex molecule of DNA consists of two polynucleotide
chains twisted around one another to form a right-handed helix in
which the bases form hydrogen bonds.
• Adenine pairs with thymine; guanine with cytosine
• A hydrogen bond is a weak bond
• The stacking of the base pairs on top of one another also contribute
to holding the strands together
• The paired bases are planar, parallel to one another, and
perpendicular to the long axis of the double helix.
Figure 06.F06A: Normal base pairs in DNA. On the left, the hydrogen bonds (dotted
lines) and the joined atoms are shown in red. (A, B) An A T base pair.
Figure 06.F06C: Normal base pairs in DNA. On the left, the hydrogen bonds (dotted
lines) and the joined atoms are shown in red. (C, D) A G C base pair.
Figure 06.F05A: Two representations of DNA illustrating the threedimensional
structure of the double helix. (A) A “ribbon diagram.”
DNA Structure
• The backbone of each strand
consists of deoxyribose sugars
alternating with phosphate groups
that link 5' carbon of one sugar to
the 3' carbon of the next sugar in
line
• The two polynucleotide strands of
the double helix run in opposite
directions. The paired strands are
said to be antiparallel
Figure 06.07: DNA molecule
showing the antiparallel
orientation of the complementary
strands.
DNA: Watson-Crick Model
3-D structure of the DNA molecule:
• Double helix forms major and minor grooves
• Diameter of the helix = 20 Angstroms
• Each turn of the helix = 10 bases = 34 Angstroms
Figure 06.05A: Illustration of DNA helix.
Part B Courtesy of Antony M. Dean,
University of Minnesota
Figure 06.05B: Computer model of DNA
helix.
DNA Replication
Watson-Crick model of DNA
replication:
• Hydrogen bonds between DNA
bases break to allow strand separation
• Each DNA strand is a template for the
synthesis of a new strand
• Template (parental) strand determines
the sequence of bases in the new strand
(daughter): complementary base pairing
rules
Figure 06.08: Watson–Crick
model of DNA replication.
DNA Replication
• In 1958 M. Meselson and
F. Stahl showed that DNA
replication is
semiconservative:
– The parental strands
remain intact and
serves as a template for
a new strand
Figure 06.09: Predictions of
semiconservative DNA replication.
Figure 6.10: The Meselson-Stahl experiment on DNA replication
The predicted result of conservative replication
Circular DNA Replication
• Autoradiogram of the intact replicating circular
chromosome of E. coli shows that
– DNA synthesis is bidirectional
– Replication starts from a single site called origin of
replication (OR)
• The region in which parental strands are separating
and new strands are being synthesized is called a
replication fork
Figure 06.12: The distinction between unidirectional and bidirectional DNA
replication.
Figure 6.11: Autoradiogram of intact replicating chromosome of E. coli
Reproduced from J. Cairns, Cold Spring Harb. Symp. Quant. Biol. 28
(1963): 43-46. © Cold Spring Harbor Laboratory Press.
Rolling Circle Replication
• Some circular DNA molecules of a number of bacterial
and eukaryotic viruses, replicate by a different mode
called rolling-circle replication.
• One DNA strand is cut by a nuclease to produce a
3'-OH extended by DNA polymerase.
• The newly replicated strand is displaced from the
template strand as DNA synthesis continues.
• Displaced strand is template for complementary
DNA strand.
Figure 06.13: Rolling-circle replication.
Replication of Linear DNA
• The linear DNA duplex in a eukaryotic chromosome
also replicates bidirectionally
• Replication is initiated at many sites along the DNA
• Multiple initiation is a means of reducing the total
replication time
Figure 06.14: Replicating DNA of D. melanogaster.
Micrograph courtesy of David S. Hogness, Department of
Biochemistry, Stanford School of Medicine
Replication of Linear DNA
• In eukaryotic cell, origins of replication are about
40,000 bp apart, which allows each chromosome to
be replicated in 15 to 30 minutes.
• Because chromosomes do not replicate
simultaneously, complete replication of all
chromosomes in eukaryotes usually takes from 5 to
10 hours.
DNA Synthesis
• One strand of the newly made DNA, leading
strand, is synthesized continuously.
• The other, lagging strand is made in small
precursor fragments = Okazaki fragments
• The size of Okazaki fragments is 1000–2000
base pairs in prokaryotic cells and 100–200
base pairs in eukaryotic cells.
Figure 06.22: Short fragments in the replication fork.
DNA vs. RNA
• DNA sugar = deoxyribose
ribose
RNA sugar =
• RNA contains the pyrimidine uracil (U) in place of
thymine (T)
• DNA is double-stranded
• RNA is single-strand
• Short RNA fragment serves as a primer to initiate
DNA synthesis at origins of replication
Figure 6.17: Differences between DNA and RNA
DNA Replication: Proteins
• Gyrase = topoisomerase II introduces a doublestranded break ahead of the replication fork and
swivels the cleaved ends to relieve the stress of
helix unwinding
• Helicase unwinds DNA at replication fork to
separate the parental strands
• Single-strand binding protein (SSB) stabilizes single
strands of DNA at replication fork
DNA Replication: Proteins
• Multienzyme complex called primosome initiates
strand synthesis by forming RNA primer
• The enzyme DNA polymerase forms the
phosphodiester bond between adjacent nucleotides
in a new DNA acid chain in 5' to 3' direction
• DNA polymerase has a proofreading function that
corrects errors in replication
DNA Replication: Proteins
• The final stitching together of the lagging strand
must require:
Removal of the RNA primer
Replacement with a DNA sequence
Joining adjacent DNA fragments
• Primer removal and replacement in E. coli is
accomplished by a special DNA polymerase (Pol I)
that removes one ribonucleotide at a time
DNA Replication: Proteins
• In eukaryotes, the primer RNA is removed as an
intact unit by a protein called RPA (replication
protein A)
• DNA ligase catalyzes the formation of the final
bond connecting the two precursor
Figure 06.15: Role of proteins in DNA replication.
Figure 6.16: DNA gyrase
Figure 6.18: Priming of DNA synthesis with an RNA segment
Figure 6.19: New DNA strand structure
Figure 6.20: Addition of nucleotides to the 3’ -OH terminus of a growing
strand
Figure 6.21: The 3’-to-5’ exonuclease activity of the proofreading function
Figure 6.23: Sequence of events in the joining of adjacent precursor
fragments in eukaryotes
Nucleic Acid Hybridization
• DNA denaturation: Two DNA strands can be
separated by heat without breaking phosphodiester
bonds
• DNA renaturation = hybridization: Two single
strands that are complementary or nearly
complementary in sequence can come together to
form a different double helix
• Single strands of DNA can also hybridize
complementary sequences of RNA
Figure 06.24: Nucleic acid hybridization.
Restriction Enzymes
• Restriction enzymes cleave duplex DNA at particular
nucleotide sequences
• The nucleotide sequence recognized for cleavage by a
restriction enzyme is called the restriction site of the
enzyme
• In virtually all cases, the restriction site of a restriction
enzyme reads the same on both strands
• A DNA sequence with this type of symmetry is called
a palindrome
Restriction Enzymes
• Many restriction enzymes all cleave their restriction
site asymmetrically—at a different site on the two
DNA strands
• They create sticky ends = each end of the cleaved site
has a single-stranded overhang that is complementary
in base sequence to the other end
• Some restriction enzymes cleave symmetrically— at
the same site in both strands
• They yield DNA fragments that have blunt ends
Restriction Enzymes
• Because of the sequence specificity, a particular
restriction enzyme produces a unique set of restriction
fragments for a particular DNA molecule.
• Another enzyme will produce a different set of
restriction fragments from the same DNA molecule.
• A map showing the unique sites of cutting of a
particular DNA molecule by restriction enzyme is
called a restriction map
Figure 6.25: DNA cleavage by the restriction enzyme BamHI
Table 6.2: Some Restriction Endonudeases, Their Sources, and Their
Cleavage Sites
Figure 06.26: Gel diagrams showing the sizes of restriction fragments produces by
digestion of a 10-kb circular DNA.
Southern Blot Analysis
• DNA fragments on a gel can be visualized by staining
with ethidium bromide, a dye that binds DNA
• DNA fragments can be isolated by cutting out the
region of the gel that contains the fragment and
removing the DNA from the gel.
• Specific DNA fragments are identified by hybridization
with a probe = a radioactive fragment of DNA or RNA
• Southern blot analysis is used to detect very small
amounts of DNA or to identify a particular DNA band
by DNA-DNA or DNA-RNA hybridization
Southern Blot Analysis
Steps in Southern blot procedure:
 DNA is digested by restriction enzymes
 DNA fragments are separated by gel
electrophoresis
 DNA is transferred from gel to hybridization
filter = blot procedure
 DNA denatured to produce single-stranded DNA
Southern Blot Analysis
• Filter is mixed with radiolabeled single-stranded DNA or
RNA probe at high temperatures that permit hybridization
= formation of hydrogen bonds between complementary
base pairs
• DNA bands hybridized to a probe are detected by X-ray
film exposure
Figure 06.27: Southern blot.
Polymerase Chain Reaction
• Polymerase chain reaction (PCR) makes possible the
amplification of a particular DNA fragment
• Oligonucleotide primers that are complementary to the
ends of the target sequence are used in repeated rounds
of denaturation, annealing, and DNA replication
• The number of copies of the target sequence doubles in
each round of replication, eventually overwhelming
any other sequences that may be present
Polymerase Chain Reaction
• Special DNA polymerase is used in PCR = Taq
polymerase isolated from bacterial thermophiles that
can withstand high temperature used in procedure
• PCR accomplishes the rapid production of large
amounts of target DNA that can then be identified
and analyzed
Figure 06.28: Polymerase chain reaction (PCR) for amplification.
DNA Sequence Analysis
• DNA sequence analysis determines
the order of bases in DNA
• The dideoxy sequencing method
employs DNA synthesis in the
presence of small amounts of
fluorescently labeled nucleotides
that contain the sugar dideoxyribose
instead of deoxyribose
Figure 06.29: Structures of
normal deoxyribose and the
dideoxyribose sugar used in
DNA sequencing.
DNA Sequencing: Dideoxy Method
• Modified sugars cause chain
termination because it lacks the 3'OH group - essential for attachment
of the next nucleotide in a growing
DNA strand
• The products of DNA synthesis are
then separated by electrophoresis.
In principle, the sequence can be
read directly from the gel.
Figure 06.30: Dideoxy method
of DNA sequencing.
DNA Sequencing: Dideoxy Method
• Each band on the gel is one base longer than the
previous band
• Each didyoxynucleotide is labeled by different
fluorescent dye
• G, black; A, green; T, red; C, purple
• As each band comes off the bottom of the gel, the
fluorescent dye that it contains is excited by laser light,
and the color of the fluorescence is read automatically
by a photocell and recorded in a computer
Figure 06.31: Fluorescence pattern obtained from a DNA sequencing gel.
Massively Parallel Sequencing
• The term massively parallel sequencing refers to
machines that can sequence millions of templates
simultaneously.
• Some of these machines can generate the equivalent of
200 human genomes of DNA sequence in a single
sequencing run.
• The trade-off is, that the massively parallel sequencers
are currently limited to read lengths of a few hundred
base pairs or less.
Figure 06.32: A method for massively parallel DNA sequencing that
captures and amplifies DNA template strands on a glass slide, and then
uses reversible terminators for sequencing.