Department of Biology
Biol: 1411
Foundations of Biology
Lecture 22
Oct 14, 2020
DNA Structure and function
Four key features of DNA structure:
1.
It is a double-stranded helix of uniform diameter
2.
It is right-handed
3.
Strands in antiparallel orientation based on 5′ and
3′ carbons of deoxyribose sugar
4.
Outer edges of nitrogenous bases are exposed in
the major and minor grooves
Base Pairing in DNA is Complementary
sugar-phosphate
backbone is antiparallel:
5′→3′ strand paired with
3′←5′ strand
strands have opposite
chemical polarity defined by
deoxyribose sugar
Hydroxyl
(OH)
Group
Phosphate
(OPO3-)
Group
Dr. Philips
Joining the nucleotides
into a DNA strand
https://www.chemguide.co.uk/organicprops/aminoacids/dna1.html
Nucleotides Are Added
to the 3′ End during DNA
Synthesis
DNA Structure and function
DNA has four important functions—double-helical structure is
essential:
• Genetic material stores genetic information—millions of
nucleotides; base sequence encodes huge amounts of
information.
• Genetic material is susceptible to mutation—a change in
information— possible through a simple alteration to a
sequence.
• Genetic material is precisely replicated in cell division—by
complementary base pairing.
• Genetic material is expressed as the phenotype—nucleotide
sequence determines sequence of amino acids in proteins
DNA Structure and function
• The structure of DNA suggested
a way in which the information
in DNA might be copied so that
it could be passed down to cells
produced in mitosis and
meiosis.
• Because of complementary base
pairing, the information is
contained in both strands; each
strand can act as a template to
make a new strand.
DNA Replication
One can imagine a system
where the double helix is
separated and each strand is
replicated
However, it was unclear
exactly how this happened
Biology exploring the diversity of life, third edition
•DNA structure suggests its mode of replication
From Watson and Crick’s 1953 paper
The Three Models of DNA Replication
• There were 3 possible ways DNA could
replicate:
• Semiconservative: each parental
strand is a template for a new strand.
• Conservative: The two parental
strands remain together in one
daughter molecule, while serving as a
template for another daughter molecule.
• Dispersive: Parent molecule is
dispersed among both strands in the
two daughter molecules.
DNA Replication
• E. coli were grown with 15N (a
heavy isotope that makes DNA
more dense), then transferred to a
medium with 14N.
• Resulting DNA densities could only
be explained by the
semiconservative model.
http://www.people.vcu.edu/~elhaij/bnfo300/13/Units/DNA/DNA-replication-part2.pdf
The Meselson–Stahl Experiment
bacteria grown
with heavy 15N
isotope
transferred to
media with light
14N isotope
Results: after one
round of DNA
replication, DNA had
intermediate weight;
after subsequent
rounds of DNA
replication, lightweight DNA
appeared with
intermediate-weight
DNA.
Interpretation of the Data
T0
Red = Heavy DNA
Green = Light DNA
T20
T40
The Meselson–Stahl Experiment
•The Meselson-Stahl experiment showed that DNA replication is
semiconservative.
 If conservative: the first generation of DNA molecules would have
been both high and low density, but no intermediate density
 If dispersive: the density of all DNA molecules in the first generation
would be intermediate, but this density would not be present in
subsequent generations; would shift closer to light
Biology exploring the diversity of life, third edition
What would have been the result if Meselson and
Stahl continued to grow the bacterial cells for an
additional 10 generations?
Would there have been any 15N/14N hybrids remaining?
The Meselson–Stahl Experiment
Intermediate
Heavy
and Light
Intermediate
100% Intermediate
and Light
50% of
each
Heavy
and Light
100% Intermediate
but with
more Light
50% of
each
25% heavy
75% light
One broad
band
The Polymerase Chain Reaction
How is DNA replicated?
3 Steps of DNA Replication:
Initiation—unwinding (denaturing) the DNA double helix and synthesizing
RNA primers
Elongation—synthesizing new strands of DNA using each of the
parental strands as templates
Termination— synthesis ends
How Is a Chromosome Replicated?
Origin of replication (ori) Proteins in the replication complex bind to a DNA sequence in ori.
Initiation of DNA replication in most prokaryotes
occurs at the single ori site
One origin of replication
means one replication
complex per chromosome
Initiation in eukaryotes involves many—up to tens
of thousands—of ori sites on each chromosome
Multiple points of origin along a given chromosome
DNA Replication: Initiation
DNA Helicase
DNA Replication: Initiation
First the DNA strands
must be separated
This is done by an
enzyme called DNA
Helicase – it separates
the two strands so the
nucleotides are no
longer base-paired
Biology exploring the diversity of life, third edition
DNA Replication: Initiation
As the strands separate a
protein called
Topoisomerase protects
the rest of the DNA
molecule from being
wound tighter
DNA Replication: Initiation
Also as the strands
separate they will want to
come back together to
their stable, base-paired
form
They are kept separate
by proteins which bind to
the single-stranded
portions of the DNA
molecule by singlestranded binding
proteins - SSBs
DNA Replication: Initiation
DNA polymerase cannot
just start adding
nucleotides.
It needs a starter
This is provided by an
enzyme called Primase,
which connects a few
complimentary RNA bases
to the template strand –
this is called an RNA
Primer
Biology exploring the diversity of life, third edition
DNA Replication: Elongation
Once an RNA primer is
in place, then the DNA
Polymerase can add
DNA bases, copying the
template strand
Each of the two strands now acts as a “template”
to synthesize a new complementary strand.
This seems strange, but
another complicating
factor is that the DNA
Polymerase only works in
one direction…
from 5’ to 3’
Biology exploring the diversity of life, third edition
OK, there are a lot of
weird issues with DNA Replication
Because the DNA
molecule is composed
of anti-parallel strands
copying also
proceeds in the
opposite directions
(remember the DNA
Pol only works
5’ to 3’)
Biology exploring the diversity of life, third edition
Biology exploring the diversity of life, third edition
Biology exploring the diversity of life, third edition
Biology exploring the diversity of life, third edition
Biology exploring the diversity of life, third edition
Biology exploring the diversity of life, third edition
Biology exploring the diversity of life, third edition
Biology exploring the diversity of life, third edition
DNA Replication: Elongation
• On the lagging strand,
synthesis is in the
opposite direction to fork
movement and requires
constant re-priming.
• Synthesis occurs
discontinuously, in a
series of fragments
called Okazaki
fragments.
• A different DNA
polymerase replaces the
primers with DNA.
• The final
phosphodiester linkage
between fragments is
catalyzed by DNA
ligase.
Biology exploring the diversity of life, third edition
DNA Strands Grow at the 3´ End
The enzyme DNA polymerase
creates a phosphodiester bond
between internal phosphate at
5′ carbon and hydroxyl group
at 3′ carbon.
Deoxyribonucleoside
triphosphate added to
3′ OH group
New nucleotides are
added to the new strand
at the 3’ end. Sequence
of added nucleotides
determined by
complementary base
pairing.
base-pairing rules
DNA Replication: Termination
Termination in eukaryotic occurs when the two
replication forks meet.
• Last primer is removed from the lagging
strand, no DNA synthesis occurs because
there is no 3′ end to extend—a singlestrand bit of DNA is left at each end.
• This is called: ----------------------------------
End-replication problem
What is this problem?
OH group at this
end
This primer will be
removed by a
different DNA poly.
AGAIN:
When the last primer is removed from the lagging strand, no DNA can be synthesized to replace
the resulting gap because there is no 3’ end to extend.
Therefore, the new chromosome has a short region of single-stranded DNA overhang at one
end. Then chromosome becomes shorter and this is a serious problem.
End-replication problem
3′
5′
So, this is a serious problem. Every time the
cell undergoes cell division, linear
chromosomes get shorter. Why haven’t our
chromosomes disappeared then?
Telomeres
The Solution:
Telomeres are repetitive sequences (TTAGGG) at the ends of
eukaryotic chromosomes. Telomeres are DNA sequences that do not
encode proteins.
Functions:
- Telomeres protect the important protein-coding DNA in the
chromosome from being lost.
- Telomeres extend the chromosome and prevent coding regions of
DNA from being cut off.
- Telomeres also prevent DNA repair mechanisms from mistakenly
joining chromosome ends.
Problem solved
End-replication problem
3′
5′
extension
lagging-strand
synthesis
Telomerase
Telomerase adds telomeres back on to
the end of the chromosome.
Some cells (bone marrow stem cells
stem cells, cancer cells) have
telomerase.
Telomerase: it has RNA
template that base-pairs with
the single-strand overhang.
Telomerase works as a DNA
polymerase to synthesize
new DNA.
Telomerase
• If too many telomeres are lost, cells undergo apoptosis.
Continuously dividing cells, such as bone marrow stem cells,
express telomerase and maintain their telomeres.
• Telomeres tend to shorten with aging; in cultured human cells,
telomeres are so short after 30 generations that the cells die.
• 85% of cancer cells express telomerase. Drugs that affect
telomerase activity are being developed to help with aging and
fight cancers
https://www.youtube.com/watch?v=TNKWgcFPHqw
https://www.youtube.com/watch?v=TEQMeP9GG6M
The Polymerase Chain Reaction