DNA - The Molecular Basis of Inheritance

James D. Watson & Francis H. Crick

In 1953 presented the double helix model of DNA
Two primary sources of information:
1. Chargaff Rule: #A#T and #G#C. A strange but
possibly meaningless phenomenon.
2. X-ray diffraction studies of Rosalind Franklin & Maurice H.
F. Wilkins

DNA Structure

Conclusion-DNA is a helical structure with distinctive

regularities, 0.34 nm & 3.4 nm.

1962: Nobel Prize in Physiology and Medicine

Watson, J.D. and F.H. Crick, Molecular Structure of
Nucleic Acids: A Structure for Deoxynucleic Acids. Nature
171 (1953), p. 738.

James D.
Watson

Francis H.
Crick

Maurice H. F.
Wilkins
What about?
Rosalind Franklin

The Structure of DNA

DNA is composed of four nucleotides,
each containing: adenine, cytosine,
thymine, or guanine.
The amounts of A = T, G = C, and
purines = pyrimidines [Chargaffs
Rule].
DNA is a double-stranded helix with
antiparallel strands [Watson and Crick].
Nucleotides in each strand are linked
by 5-3 phosphodiester bonds
Bases on opposite strands are linked
by hydrogen bonding: A with T, and G
with C.

The Basic Principle: Base Pairing to a Template Strand

The relationship between structure and function is manifest in the

double helix
Since the two strands of DNA are complementary each strand acts as a
template for building a new strand in replication
In DNA replication, the parent molecule unwinds, and two new daughter
strands are built based on base-pairing rules
5 end
Hydrogen bond

3 end

1 nm
3.4 nm

3 end
0.34 nm

5 end

DNA replication
The parent molecule unwinds, and two new
daughter strands are built based on basepairing rules
T

A
G

(a) The parent molecule has two

complementary strands of DNA.
Each base is paired by hydrogen
bonding with its specific partner,
A with T and G with C.

(b) The first step in replication is

separation of the two DNA
strands.

(c) Each parental strand now

serves as a template that
determines the order of
nucleotides along a new,
complementary strand.

(d) The nucleotides are connected

to form the sugar-phosphate
backbones of the new strands.
Each daughter DNA
molecule consists of one parental
strand and one new strand.

DNA Replication

DNA must replicate during each cell division

3 alternative models for DNA replication were hypothesized:
Semiconservative replication
Conservative replication
Dispersive replication

Semi-conservative

Conservative

Dispersive

Meselson-Stahl Experiments

Labeled the nucleotides of

old strands with a heavy
isotope of nitrogen (15N),
new nucleotides were
indicated by a lighter isotope
(14N).
The first replication in the
14N medium produced a
band of hybrid (15N-14N)
DNA, eliminating the
conservative model.
A second replication
produced both light and
hybrid DNA, eliminating the
dispersive model and
supporting the
semiconservative model.

Bacteria
cultured in
medium
containing

Bacteria
transferred to
medium
containing

15

14

DNA sample
centrifuged
after 20 min
(after first
replication)

DNA sample
centrifuged
after 40 min
(after second
replication)
First replication

Conservative
model

Semiconservative
model

Dispersive
model

Less
dense

More
dense

Second replication

DNA Replication is Semi-conservative

Each 2-stranded
daughter molecule is
only half new
One original strand was
used as a template to
make the new strand

DNA Replication
The copying of DNA is remarkable in its speed and accuracy
Involves unwinding the double helix and synthesizing two
new strands.
More than a dozen enzymes and other proteins participate
in DNA replication
The replication of a DNA molecule begins at special sites
called origins of replication, where the two strands are
separated

Origins of Replication
A eukaryotic chromosome may have hundreds or
even thousands of replication origins
Origin of replication

1 Replication begins at specific sites

where the two parental strands
separate and form replication
bubbles.

Parental (template) strand

Daughter (new) strand

Bubble

0.25 m

Replication fork

2 The bubbles expand laterally, as

DNA replication proceeds in both
directions.
3 Eventually, the replication
bubbles fuse, and synthesis of
the daughter strands is
complete.

Two daughter DNA molecules

(a) In eukaryotes, DNA replication begins at many sites along the giant
DNA molecule of each chromosome.

(b) In this micrograph, three replication

bubbles are visible along the DNA of
a cultured Chinese hamster cell (TEM).

Mechanism of DNA Replication

DNA replication is catalyzed by

DNA polymerase III which needs
an RNA primer
DNA polymerase III cannot
initiate the synthesis of a
polynucleotide, they can only
add nucleotides to the 3 end
The initial nucleotide strand is an
RNA primer
RNA primase synthesizes primer
on DNA strand
DNA polymerase adds
nucleotides to the 3 end of the
growing strand

DNA polymerase III adds

nucleotides to primer

DNA polymerase I degrades

the RNA primer and
replaces it with DNA

Mechanism of DNA Replication

Nucleotides are added by complementary base pairing with the template strand
DNA always reads from 5 end to 3 end for transcription replication
During replication, new nucleotides are added to the free 3 hydroxyl on the
growing strand
The nucleotides (deoxyribonucleoside triphosphates) are hydrolyzed as added,
releasing energy for DNA synthesis.
The rate of elongation is about 500 nucleotides per second in bacteria and 50
per second in human cells
New strand
5 end

Template strand
3 end

5 end

3 end

Sugar
Phosphate

Base

3 end

DNA polymerase
Pyrophosphate3 end

Nucleoside
triphosphate

5 end

5 end

The Mechanism of DNA Replication

DNA synthesis on the leading

strand is continuous
Only one primer is needed for
synthesis of the leading strand
The lagging strand grows the
same general direction as the
leading strand (in the same
direction as the Replication
Fork). However, DNA is made
in the 5-to-3 direction
Therefore, DNA synthesis on
the lagging strand is
discontinuous
For synthesis of the lagging
strand, each fragment (Okazaki)
must be primed separately, then
DNA fragments are sythesized
and subsequently ligated
together

3
5

Parental DNA

Leading strand

5
3

Okazaki
fragments
Lagging strand
3
5
DNA pol III

Template
strand

Leading strand
Lagging strand

Template
strand

DNA ligase

Overall direction of replication

Mechanism of DNA Replication

Many proteins assist in DNA replication

DNA helicases unwind the double helix, the template strands are stabilized by
other proteins
Single-stranded DNA binding proteins make the template available
RNA primase catalyzes the synthesis of short RNA primers, to which
nucleotides are added.
DNA polymerase III extends the strand in the 5-to-3 direction
DNA polymerase I degrades the RNA primer and replaces it with DNA
DNA ligase joins the DNA fragments into a continuous daughter strand
Overall direction of replication

Leading
strand

DNA pol III

Replication fork
Primase
DNA pol III
Primer

3
Parental DNA

Lagging
strand

Leading
Origin of replication
strand

Lagging
strand

Lagging
strand

Leading
strand

OVERVIEW

DNA ligase
DNA pol I
3
5

Enzymes in DNA replication

Helicase unwinds
parental double helix

DNA polymerase III

binds nucleotides
to form new strands

Binding proteins
stabilize separate
strands

Primase adds
short primer
to template strand

DNA polymerase I
(Exonuclease) removes
RNA primer and inserts
the correct bases

Ligase joins Okazaki

fragments and seals
other nicks in sugarphosphate backbone

Replication
3
3

5
3
5

Helicase protein binds to DNA sequences called

origins and unwinds DNA strands.
Binding proteins prevent single strands from rewinding.
Primase protein makes a short segment of RNA
complementary to the DNA, a primer.

Replication
Overall direction
of replication

3
3

5
3
5

3
5

DNA polymerase III enzyme adds DNA nucleotides

to the RNA primer.

Replication
Overall direction
of replication

3
5

5
3
5

3
5

DNA polymerase proofreads bases added and

replaces incorrect nucleotides.

Replication
Overall direction
of replication

3
3

5
3
5

Leading strand synthesis continues in a

5 to 3 direction.

3
5

Replication
Overall direction
of replication

3
3

Okazaki fragment

3
5

Leading strand synthesis continues in a

5 to 3 direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.

3
5

Replication
Overall direction
of replication

3
3

5
Okazaki fragment

3
5

3
5

Leading strand synthesis continues in a

5 to 3 direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.

3
5

Replication
Overall direction
of replication

3
3

5
Okazaki fragment

3
5

3 5

Leading strand synthesis continues in a

5 to 3 direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.

3
5

Replication
3
5

3
5
3
5

3 5

3 5

3
5

Leading strand synthesis continues in a

5 to 3 direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.

Replication
3
5

3
5
3
5

35

35

3
5

Leading strand synthesis continues in a

5 to 3 direction.
Discontinuous synthesis produces 5 to 3 DNA
segments called Okazaki fragments.

Replication
3
5

3
5
3
5

35

35

3
5

Exonuclease activity of DNA polymerase I

removes RNA primers.

Replication
3
3
5
3
5

35

3
5

Polymerase activity of DNA polymerase I fills the gaps.

Ligase forms bonds between sugar-phosphate backbone.

Replication Fork Overview

Overall direction of replication

Leading
strand

Lagging
strand

DNA pol III

Replication fork

3
Parental DNA

Primase
Primer

Lagging
strand

Lagging
Leading
Origin of replication
strand
strand

OVERVIEW

Leading
strand

DNA ligase
DNA pol I

DNA pol III

3
5

Other Proteins That Assist DNA Replication

Helicase, topoisomerase, single-strand binding
protein are all proteins that assist DNA replication

Proofreading
Mistakes during the initial pairing of template
nucleotides and complementary nucleotides occur at
a rate of one error per 100,000 base pairs.
DNA polymerase proofreads each new nucleotide
against the template nucleotide as soon as it is
added and can correct errors
If there is an incorrect pairing, the enzyme removes
the wrong nucleotide and then resumes synthesis.
Mismatched nucleotides that are missed by DNA
polymerase or mutations that occur after DNA
synthesis is completed can often be repaired

Mutations
Mismatch repair: wrong inserted base can be removed
Excision repair: DNA may be damaged by chemicals,
radiation, etc. Mechanism to cut out and replace with
correct bases
Each cell continually monitors and repairs its genetic
material, with 100 repair enzymes known in E. coli and
more than 130 repair enzymes identified in humans.
The final error rate is only one per ten billion nucleotides
Because the human genome is so large, even at this
rate, mutations add up. Each of us probably inherited 34 mutations!

Proofreading and Repairing DNA

DNA polymerases
proofread newly made
DNA, replacing any
incorrect nucleotides
In mismatch repair of DNA,
repair enzymes correct
errors in base pairing
In nucleotide excision DNA
repair nucleases cut out
and replace damaged
stretches of DNA

1 A thymine dimer
distorts the DNA molecule.
2 A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
Nuclease

DNA
polymerase

3 Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA
ligase

4 DNA ligase seals the

Free end of the new DNA
To the old DNA, making the
strand complete.

DNA repair

Accuracy of DNA Replication

The chromosome of E. coli bacteria contains
about 5 million bases pairs
Capable of copying this DNA in less than an hour

The 46 chromosomes of a human cell contain

about 6 BILLION base pairs of DNA!!
Printed one letter (A,C,T,G) at a timewould fill up
over 900 volumes of Campbell.
Takes a cell a few hours to copy this DNA
With amazing accuracy an average of 1 per
billion nucleotides

Replicating the Ends of DNA Molecules

The ends of eukaryotic chromosomal DNA get
shorter with each round of replication
5

Leading strand
Lagging strand

End of parental
DNA strands
3
Last fragment

Previous fragment

RNA primer
Lagging strand

3
Primer removed but
cannot be replaced
with DNA because
no 3 end available
for DNA polymerase

Removal of primers and

replacement with DNA
where a 3 end is available
5

3
Second round
of replication
5
New leading strand 3
New lagging strand 5

3
Further rounds
of replication
Shorter and shorter
daughter molecules

Telomeres
Eukaryotic chromosomal DNA molecules
have at their ends nucleotide sequences,
called telomeres, that postpone the erosion of
genes near the ends of DNA molecules

1 m

Telomerases
If the chromosomes of germ cells became
shorter in every cell cycle essential genes
would eventually be missing from the gametes
they produce
An enzyme called telomerase catalyzes the
lengthening of telomeres in germ cells