Genetics chapter 7 dna structure and replication

DNA:
The Chemical Nature of the Gene

The remarkable
stability of DNA
makes the extraction
and analysis of DNA
from ancient remains
possible, including
Neanderthal bones
that are more than
30,000 years old!

More on this later…
More on this later…

The Genetic Material
MUST..
• Store information
• Information can vary
but be stable
• Replicate
• DNA replication
• Encode the phenotype
• Govern expression:
gene function

BEFORE DNA WAS
IDENTIFIED, WHAT WHERE
WE LOOKING FOR?

5 Features of Hereditary Material
1. Localized to the nucleus,
component of chromosomes
2. Present in stable form in cells
3. Sufficiently complex to contain
information needed for
structure, function,
development, and
reproduction of an organism

5 Features of Hereditary Material, continued
4. Able to accurately replicate
itself so that daughter cells
contain the same information
as parent cells
5. Mutable, undergoing a low
rate of mutations that
introduces genetic variation
and serves as a foundation
for evolutionary change

DNA: The Early Age
• Friedrich Meischer (1869):
extracted substance from
white blood cells
• Slightly acidic and phosphorus rich
• DNA and protein

Friedrich Meischer

• Kossel (1901): DNA contains
A, G, C, and T
• Phoebus Albert Levene
(1910): Tetranucleotide Theory

Ludwig Karl Martin
Leonhard Albrecht Kossel
(16 September 1853 – 5 July
1927)

• DNA is made of nucleotides
• Not variable

• Erwin Chargaff (11 August
1905 – 20 June 2002)

Phoebus Aaron
Theodore Levene, M.D.
(25 February 1869 – 6
September 1940)

Chargaff’s Rules
In all organisms, the nucleotide bases are found in specific
proportions.
• Chargaff’s rules are based on two observations.

– The amount of A, C, G, and T varies from
species to species.
– In each species, the amount of A is equal to T
and the amount of G is equal to C.

Pneumococcus & Bacteriophage
Experiment Series

EVIDENCE THAT DNA IS
THE MOLECULE
RESPONSIBLE FOR
CONVEYING HEREDITARY
CHARACTERISTICS
Is it DNA, RNA, lipids, carbohydrates, etc.?

The Transformation Factor
• Frederick Griffith identified
two strains of Pneumococcus:
S, which caused fatal
pneumonia in mice, and R,
which did not
• S = smooth = protective
capsule

• A single nucleotide change
can convert the S (smooth)
strain into R (rough) the strain
• R has a mutant allele on the
polysaccharide gene

Griffith’s Experimental Results
• Mice infected with strain
SIII developed pneumonia
and died
• Mice infected with strain
RII or with heat-killed strain
SIII survived
• Mice infected with heatkilled strain SIII and live
strain RII developed
pneumonia and died – livetype SIII bacteria were
recovered from the mice

Avery, MacLeod, and McCarty

• What is the Hereditary Molecule?
• Avery, MacLeod, and McCarty used heat-killed SIII
bacteria and live RII bacteria and infected mice
• The extract of heat-killed SIII bacteria was divided
into aliquots and treated to destroy either DNA,
RNA, proteins, or lipids and polysaccharides
• All aliquots killed the mice except the one with the
DNA destroyed

DNA Is the Hereditary Molecule!
• Are we sure? Replication and verification is an
important part of the scientific process….
• Hershey and Chase, in 1952, showed that DNA is
responsible for bacteriophage infection of bacteria
cells

Bacteriophages
(phages) are
viruses that infect
bacteria

Awesome
Awesome
Phage Lab
Phage Lab
here at
here at
VCU! 
VCU! 

Phage Infection of Bacteria
• Phages must infect bacterial
hosts to reproduce
• Infection begins when the
phage injects DNA into the
bacterial cell and leaves its
protein shell on the surface
(ghost!)
• The phage DNA replicates in
the bacterium and produces
proteins that are assembled
into progeny phages – these
are released by lysis of the
host cell

Hershey and Chase Experiments
• Distinguish DNA and protein by their sulfur and
phosphorous content
• Proteins contain large amounts of sulfur but almost no
phosphorus
• DNA contains large amounts of phosphorus but no sulfur

• Hershey and Chase separately labeled either phage
proteins (with 35S) or DNA (with 32P) and then traced
each radioactive label in the course of infection

Conclusion
• These results demonstrate that phage DNA, not
phage protein, is transferred to host bacterial cells
and directs synthesis of phage DNA and proteins,
the assembly of progeny phage particles, and
ultimately lysis of infected cells.
• Furthermore, this experiment demonstrated that the
transformation factor previously identified by Griffith
was DNA
• It also showed that Avery, MacLeod and McCarty
were correct in concluding that DNA is the hereditary
material.

DNA is the heritable
material…
BUT WHAT IS THE
STRUCTURE OF DNA?

1951 - Recognize Rosalind Franklin, Yo!

7.2 The DNA Double Helix Consists of Two
Complementary and Antiparallel Strands
• Watson and Crick’s model
of the secondary structure
of DNA shows that it is fairly
simple in structure
• It is composed of four kinds
of nucleotides, joined by
covalent phosphodiester
bonds with two
polynucleotide chains that
come together to form a
double helix

Nucleotides have a polarized
structure

Phosphate end: 5’cabon (five prime end)
OH in sugar : 3’carbon (three prime end)

Two Types of DNA Bases

• Pyrimidines, thymine or
cytosine, have a single ring,
and purines, adenine or
guanine, have a double ring
• Deoxynucleotide
monophosphates that are
part of a polynucleotide chain
have single phosphates and
are called dNMPs, where N
refers to any of the four
bases
• Deoxynucleotide
triphosphates, dNTPs, are
not part of a polynucleotide
chain

dNTPs are
dNTPs are
recruited by DNA
recruited by DNA
polymerase
polymerase

Pyrophosphate
Pyrophosphate
group discarded
group discarded

The diameter results from the fact that each complementary base pair (A
and T or G and C) is 20Å wide

Base-pair stacking creates gaps
between the sugar phosphate
backbones that partially expose
the nucleotides

The major groove,
approximately 12Å wide,
alternates with the minor
groove, approximately
6Å wide
These grooves are regions
where DNA binding proteins
can make direct contact with
nucleotides

Three Attributes of DNA
Replication Shared by All
Organisms
1. Each strand of the
parental DNA molecule
remains intact during
replication
2. Each parental strand
serves as a template for
formation of an
antiparallel,
complementary daughter
strand
3. Completion of replication
results in the formation of
two identical daughter
duplexes composed of
one parental and one
daughter strand

7.3 DNA Replication Is Semiconservative and
Bidirectional
• The general mechanism
of DNA replication is the
same in all organisms
• As organisms diverged
and became more
complex, some
differences did develop
in the replication
proteins and enzymes
• These subtle differences
are why antibiotics stop
replication in bacteria,
but not us!

Origin and direction of replication
• Is origin single or multiple?
• Does it start at random or specific
location?
• Is it unidirectional or bidirectional?

We can see DNA replicating!
• John Cairns grew
bacteria in a medium
containing 3H-thymine
• He extracted the
bacterial chromosomes
during replication and
placed them on x-ray film
(radioactive decay of 3H
is slow, this took several
months!)
• The autoradiographs
showed dark lines that
revealed the pattern of
replicating DNA
molecules – these were
called theta structures
(θ structures)

Evidence of Bidirectional DNA Replication
• In bidirectional DNA replication, new DNA is synthesized in both
directions from the single origin, creating an expanding replication
bubble
• At each end of the replication bubble is a replication fork; replication is
complete when the replication forks meet

41
Genetics Analysis: An Integrated Approach

Copyright © 2012 Pearson Education Inc.

The location of the origin of
replication and replication
terminus regions on opposite
sides of the E. coli
chromosome.

Additional Support for Bidirectional
Replication
• Biochemical studies of DNA
polymerase of E. coli show that the
polymerase can incorporate about
1000 nucleotides per second into the
new DNA strand
• At this rate of synthesis, the entire
genome can be replicated in
approximately 33 minutes
• This corresponds well with the
generation time of
E. coli; unidirectional synthesis would
require twice as much time!

Multiple Replication Origins in Eukaryotes
• Autoradiograph analysis
shows multiple origins of
replication on eukaryotic
chromosomes
• Large eukaryotic genomes
contain thousands of origins
of replication separated by
40,000 to 50,000 base pairs
• The human genome
contains more than 10,000
origins!
• DNA replication rate varies
among different types of
cells

Multiple origins of replication on a single
chromosome from Drosophila melanogaster

Bacterial Replication Origins
• Replication origins have sequences that attract replication enzymes
• The origin of replication sequence of E. coli is called oriC, and it contains
about 245 bp of A-T rich DNA
• The origin is divided into three 13-bp sequences followed by four 9-bp
sequences

Replication Initiation in Bacteria
• Replication in E. coli requires that
replication-initiating enzymes locate and
bind to oriC consensus sequences
• Enzymes DnaA, DnaB, DnaC bind at oriC
and initiate DNA replication
• DnaA
•

Binds the 9-mer, bends the DNA,

•

breaks hydrogen bonds in the A-T rich 13mer sequences

• DnaB is a helicase
•

uses ATP energy

•

separate the strands and unwind the helix

•

DnaB is carried to the DNA helix by DnaC

• Single-stranded binding protein (SSBP)
•

Prevents reannealing of unwound strands

Topoisomerases
Topoisomerases

Unwinding of circular chromosomes will create torsional stress, potentially
leading to supercoiled DNA
Enzymes called topoisomerases catalyze controlled cleavage and
rejoining of DNA that prevents over-winding

Eukaryote Replication Origins
• Saccharomyces cerevisiae (yeast) has the most fully characterized originof-replication sequences
• The multiple origins of replication are called autonomously replicating
sequences (ARS)
• These sequences help orient the replication machinery before replication
begins!

RNA Primers Are Needed for
DNA Replication
• DNA polymerase
elongates DNA strands
by adding nucleotides to
the 3′ end of a preexisting strand
• They cannot initiate
DNA strand synthesis
on their own
• RNA primers are
needed; these are
synthesized by a
specialized RNA
polymerase called
primase

DNA Polymerase III
• In E. coli, daughter DNA strands
are synthesized by the DNA
polymerase III (pol III)
holoenzyme
• Holoenzyme refers to a
multiprotein complex in which a
core enzyme is associated with
the additional components
needed for full function
• The replisome is found at each
replication fork and contains two
copies of pol III

ANIMATION: DNA Replication-4


Leading and Lagging Strand Synthesis
• Leading strand:
pol III synthesizes
one daughter
strand continuously
in the same
direction as fork
progression
• Lagging strand:
pol III elongates the
daughter strand
discontinuously, in
the opposing
direction to fork
progression, via
short segments
(Okazaki
fragments)

RNA Primer Removal and Okazaki Fragment
Ligation

Simultaneous Synthesis of Leading and
Lagging Strands
• Each replisome complex
carries out replication of the
leading and lagging strand
simultaneously
• The DNA pol III holoenzyme
contains 11 protein subunits,
with the two pol III core
polymerases each tethered to
a different copy of the tau (τ)
protein
• The tau proteins are joined to
a protein complex called the
clamp loader; two additional
proteins form the sliding
clamp

The Sliding Clamp

• The sliding clamp can close
around the double-stranded
DNA during replication
• It has a “doughnut hole” of
about 35Å, into which the
DNA fits
• The sliding clamp anchors
the DNA pol III core enzyme
to the template
• It is required for the high
level of pol III activity

ANIMATION: Molecular Model of DNA Replication


FOUNDATION
FOUNDATION
FIGURE 7.24
FIGURE 7.24
The Trombone
Model of DNA
Replication

http://www.yo
utube.com/w
atch?
v=5VefaI0Lrg
E&feature=rel
ated

DNA Proofreading
• DNA replication is very
accurate, mainly because
DNA polymerases
undertake DNA
proofreading, to correct
occasional errors
• Errors in replication occur
about one every billion
nucleotides in E. coli
• Proofreading ability of DNA
polymerase enzymes is due
to a 3′ to 5′ exonuclease
activity

You should know the
You should know the
functions of these
functions of these
molecules; spend less
molecules; spend less
time on naming
time on naming
details!
details!

Telomeres
• The leading strand of linear chromosomes can be replicated to the end
• The lagging strand requirement for a primer means that lagging strands
cannot be completely replicated
• This problem is resolved by repetitive sequences at the ends of
chromosomes, called telomeres
• These repeats ensure that incomplete chromosome replication does not
affect vital genes

Telomerase
• Telomeres are synthesized
by the ribonucleoprotein
telomerase
• Blackburn, Greider, and
Szostak received the 2009
Nobel Prize for the discovery
of telomeres and telomerase
• The RNA in telomerase is
complementary to the
telomere repeat sequence
and acts as a template for
addition of DNA

Telomerase Function
• The template RNA of
telomerase allows new
DNA replication, to lengthen
the telomere sequences
• Once telomeres are
sufficiently elongated, the α
polymerase synthesizes
additional RNA primers
• New DNA replication then
fills out the chromosome
ends
• Telomere sequences in
most organisms are quite
similar

Importance of Telomerase Activity
• Mice that are homozygous for
loss-of-function mutations of
the TERT (telomerase reverse
transcriptase) gene give rise to
developmental defects
• The defects are first observed
in the fourth and fifth
generations, due to loss of
telomere length with each
generation
• By the sixth generation,
shortening of chromosomes is
critical and apoptosis is
induced

Left, 48-week-old TERT-ER mouse with activated telomerase. Right, 35week-old TERT-ER mouse, not treated. (Dana-Farber Cancer Institute)

Chromosome spreads showing telomere elongation after telomerase activation
68
(right). DNA in blue, telomeres in red. (Dana-Farber Cancer Institute)

Telomeres, Aging, and Cancer
• Telomere length is important for
chromosome stability, cell longevity,
and reproductive success
• Telomerase is active in germ-line
cells and some stem cells in
eukaryotes
• Differentiated somatic cells and cells
in culture have virtually no
telomerase activity; such cells have
limited life spans (30 to 50 cell
divisions)
• Hayflick Limit: limitation on the
growth of most cells grown in culture

paw.princeton.edu

www.scientificamerican.com

Werner Syndrome
• Telomerase inactivity is
associated with normal
aging of cells
• A condition known as
Werner syndrome
causes early onset of
some features of aging
• A mutation in RECQL2,
a gene encoding a
helicase required for
telomerase activity, is
the cause of Werner
syndrome

Dyskeratosis Congenita
• Dyskeratosis congenita
is a disorder associated
with a loss of function of
a gene, DKC1, that
encodes a protein
needed for normal
telomerase function
• Patients with this
disorder have skin and
nail abnormalities, loss
of vision and hearing,
and abnormalities of
blood cell formation

Abnormal Reactivation of Telomerase
Activity
• Telomerase is normally
turned off in somatic cells
• Reactivation of telomerase
can lead to aging cells that
continue to proliferate, a
feature of many types of
cancer
• TERT reactivation is one
of the most common
mutations in cancers of all
types

7.5 Molecular Genetic Analytical Methods Make
Use of DNA Replication Processes
• Molecular biologists have used their understanding
of DNA replication to develop new methods of
molecular analysis
• Two widely used methods include polymerase chain
reaction (PCR) and dideoxynucleotide DNA
sequencing

The Polymerase Chain Reaction
• The polymerase chain
reaction (PCR) is an
automated version of DNA
replication that produces
millions of copies of a short
target DNA segment
• There are numerous
applications of PCR
•

-Genetic testing, oncogenes,
detection of disease (HIV, etc),
forensics (genetic fingerprinting),
research (sequencing, cloning,
gene expressions, phylogenetic
analysis, rapid DNA production)

• PCR reactions are carried out
in small volumes (less than
100 µ l)

The Process of PCR
• PCR uses two DNA
sequences called PCR
primers that provide a starting
point for Taq polymerase to
add nucleotides
• The PCR primers define the 5′
and 3′ boundaries of the
replication products
• PCR is composed of three
steps that result in exponential
amplification of large numbers
of the target DNA

Components of PCR
• PCR requires
• A double-stranded DNA template
containing the target sequence to
be amplified
• A supply of the four DNA
nucleotides
• A heat-stable DNA polymerase
• Two different single-stranded DNA
primers
• A buffer solution
• The most commonly used DNA
polymerase, Taq, is isolated from
Thermus aquaticus, which occurs
naturally in hot springs

http://microbewiki.kenyon.edu/index.php/Thermus_aquaticus*

Steps of PCR
1.

Denaturation: the reaction is
heated to ∼ 95⁰ C to
denature the DNA into single
strands

2.

Primer annealing: the reaction
temperature is reduced to ∼ 45-68⁰
C to allow primers to hybridize
to their complementary sequences
in the target DNA

3.

Primer extension: the reactions
temperature is raised to 72⁰ C to
allow Taq polymerase to synthesize
DNA

Limitations of PCR
1. Some knowledge of the target DNA sequences is
required in order to determine primer sequences
2. Amplification products longer than 10 to 15 kb are
difficult to produce
•

Despite the limitations, PCR is a practical way to
obtain large quantities of DNA from a particular
gene for molecular analysis

Separation of PCR Products
• Amplified DNA fragments are
separated from the rest of the
reaction mixture by gel
electrophoresis and
visualized by ethidium
bromide staining
• PCR product sizes are
measured in base pairs (bp)
• Differences in the size of
DNA amplified by a pair of
primers are related to the
amount of DNA between the
primers

http://passel.unl.edu

What do we do with PCR Products?
• Genetic testing
• Human consoling: amniocentesis
• Disease detection: viruses
• Tissue typing: organ transplantation
• Cancer: oncogenes

• Forensic applications

http://theinvestigation.yolasite.com/pc
r-testing.php

• DNA Fingerprinting: VNTRs (next!)

• RESEARCH!
• Hybridization probes, DNA sequencing, clonining,
phylogenetic analysis of ancient DNA (coming soon!) &
MORE!!

ANIMATION: Polymerase Chain Reaction (PCR)-1


Variable Number Tandem Repeats
•

A Variable Number Tandem Repeat
(or VNTR) is a location in a genome
where a short nucleotide sequence is
organized as a tandem repeat. These
can be found on many chromosomes,
and often show variations in length
between individuals. Each variant
acts as an inherited allele, allowing
them to be used for personal or
parental identification. Their analysis
is useful in genetics and biology
research, forensics, and DNA
fingerprinting.

• Codominant
fingerprint!

Different
father

Adopted

Within the VNTRs there are sites where an enzyme can cut the DNA,
and the location of these sites also varies from person to person.
Cutting with the enzyme will lead to DNA fragments of different
lengths, which are called Restriction Fragment Length Polymorphisms
(RFLPs).
http://www.scq.ubc.ca/a-brief-tour-of-dna-fingerprinting/

Variable Number Tandem Repeats
•

VNTRs have variable numbers of repeats of DNA of up to 20 bp in length

•

VNTRs are inherited, as are other types of alleles, and can be detected through PCR

Hereditary
transmission of
VNTR alleles
follows a
codominant pattern

Dideoxynucleotide DNA Sequencing
• The ultimate description of a DNA
molecule is its precise sequence
of bases
• The first DNA-sequencing
protocols were developed by
Maxam and Gilbert, and another
by Sanger
in 1977
• The Sanger (dideoxynucleotide)
Method was most amenable to
automation and is the method of
choice today

Frederick Sanger
Born 1918, won two Nobel Prizes

Sanger Sequencing
• Dideoxynucleotide DNA
sequencing (dideoxy
sequencing) uses DNA
polymerase to replicate new
DNA from a single-stranded
template
• The four standard
deoxynucleotide bases
(dNTPs) are present in large
amounts
• Each reaction contains a small
amount of one
dideoxynucleotide (ddNTP),
which lacks a 3′-OH group
• Didexoy = TWO
deoxygenated sites!

The Principle of Sanger Sequencing
• When replication
starts, most
fragments will
incorporate a
dNTP and
continue
replication
• BUT, whenever a
ddNTP is
incorporated into
the product DNA
molecule,
replication ceases

Sanger Sequencing
• A separate reaction is
carried out for A, T, G,
and C, using the
corresponding small
amount of ddNTP
• Each reaction tube
produces a series of
partial DNA molecules,
each of which ends with
that nucleotide
• All four reactions must be
run side by side on a gel
in order to determine the
complete sequence

Visualization of DNA Sequence
• After the reactions are
complete, the reactions
are run side by side on a
gel
• Autoradiograph bands are
visualized by labeling the
5’ primers with radioactive
isotopes or fluorescent
tags (end-labeling)
• The shortest bands are
the DNA products closest
to the primer and these
travel fastest on the gel;
the gel is read from the
bottom up, all four lanes
together

Automated DNA Sequencing
• Automated DNA sequencers use a
single reaction for each DNA
sequence, in which all four ddNTPs
are included
• Each ddNTP is labeled with a unique
fluorescent marker
• The DNA is synthesized, and a
mixture of fragments is produced and
run on a DNA gel
• The fluorescent label on each ddNTP
has a different wavelength, and a laser
light excites the fluorescent tag on
each fragment as it passes
• The fluorescence pattern produced
shows the sequence of the DNA

Genetics chapter 7 dna structure and replication

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