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Mechanisms of Gene Mutation
Lecture 7
Dr. Attya Bhatti
Molecular basis of gene mutations

Are of two types
1. Spontaneous mutations
2. Induced mutations

Can be analyzed by Molecular Genetic
Techniques.
Spontaneous mutations

Arise from a variety of sources,
Errors in DNA replication,
2. Spontaneous lesions,
3. Transposable genetic elements.
1.
Errors in DNA Replication
Pairing between the normal (keto) forms of the bases.
Errors in DNA Replication
Mismatched bases. (a) Mispairs resulting from rare tautomeric
forms of the pyrimidines; (b) mispairs resulting from rare
tautomeric forms of the purines.
A keto structure occurs when the hydrogen atom bonds to a nitrogen
atom within the ring. An enol structure occurs when the hydrogen atom
bonds to an nearby oxygen atom that sticks out from the ring. These two
types of structures are known as tautomers.
Errors in DNA Replication
Results in
1. Transitions
2. Transversions
3. Frameshift mutations
4. Deletions and duplications

Spontaneous lesions


Result from depurination
and deamination.
Depurination, the more
common of the two,
consists
of
the
interruption
of
the
glycosidic bond between
the nitrogenous base and
deoxyribose and the
subsequent loss of a
guanine or an adenine
residue from the DNA.
Fig: The loss of a purine
residue (guanine) from a single
strand of DNA. The sugarphosphate backbone is left
intact.
Spontaneous lesions


The
deamination
of
cytosine yields uracil.
Unrepaired
uracil
residues will pair with
adenine in replication,
resulting
in
the
conversion of a G–C pair
into an A–T pair (a
GC → AT transition).
Fig: Deamination of
cytosine
and
(b)
methylcytosine.
(a)
5-
Spontaneous mutations and human diseases
Disorders are due to deletions or duplications involving
repeated sequences.
 For example, mitochondrial encephalomyopathies are a
group of disorders affecting the central nervous
system or the muscles (Kearns-Sayre syndrome).

Fig: Sequences of wild-type (WT) mitochondrial DNA and
deleted DNA (KS) from a patient with Kearns-Sayre syndrome.
The 13-base boxed sequence is identical in both WT and KS
and serves as a breakpoint for the DNA deletion. A single base
(boldface type) is altered in KS, aside from the deleted
segment.
Spontaneous mutations and human diseases
Genetic diseases due to the expansion of a three-basepair repeat.
 For example; Fragile X syndrome

Fig: Expansion of the CGG triplet in the FMR-1 gene seen in the fragile
X syndrome. Normal persons have from 6 to 54 copies of the CGG
repeat, whereas members of susceptible families display an increase
(premutation) in the number of repeats: normally transmitting males
(NTMs) and their daughters are phenotypically normal but display from
50 to 200 copies of the CGG triplet; the number of repeats expands to
some 200 to 1300 in those showing full symptoms of the disease.
Induced mutations


1.
2.
3.
Introduction of mutations by mutagens.
Mutagens induce mutations by at least three
different mechanisms.
Incorporation of base analogs.
Specific mispairing.
Base damage.
Incorporation of base analogs
Fig: Alternative pairing possibilities for 5-bromouracil (5-BU). 5BU is an analog of thymine that can be mistakenly incorporated
into DNA as a base. It has a bromine atom in place of the methyl
group. (a) In its normal keto state, 5-BU mimics the pairing
behavior of the thymine that it replaces, pairing with adenine. (b)
The presence of the bromine atom, however, causes a relatively
frequent redistribution of electrons, so that 5-BU can spend part
of its existence in the rare ionized form. In this state, it pairs
with guanine, mimicking the behavior of cytosine and thus
inducing mutations in replication.
Specific mispairing
Some mutagens are not incorporated into the DNA but
instead alter a base, causing specific mispairing.
 E.g
Certain
alkylating
agents,
such
as
ethylmethanesulfonate (EMS) and Intercalating agents
such as proflavin, acridine orange.

Fig: Intercalating agents. (a) Structures of the common
agents proflavin, acridine orange, and ICR-191. (b) An
intercalating agent slips between the nitrogenous bases
stacked at the center of the DNA molecule. This occurrence
can lead to single-nucleotide-pair insertions and deletions.
Base damage

A large number of mutagens damage one or more bases,
so no specific base pairing is possible.

The result is a replication block, because DNA synthesis
will not proceed past a base that cannot specify its
complementary partner by hydrogen bonding.
Base damage
•Ionizing radiation can cause breakage of the Nglycosydic bond, leading to the formation of AP sites,
and can cause strand breaks that are responsible for
most of the lethal effects of such radiation.
•AP site
Apurinic or apyrimidinic site resulting from the loss of a
purine or pyrimidine residue from the DNA
Relation between mutagens and
carcinogens

157 of 175 known carcinogens (approximately 90
percent) are mutagens.
Induced mutations and human cancer
Ultraviolet light and aflatoxin B1 have long been
suspected of causing skin cancer and liver cancer,
respectively.
 DNA sequence analysis of mutations in a human cancer
gene has provided direct evidence of their involvement.
 p53 is one of a number of tumor-suppressor genes that
encode proteins that suppress tumor formation.
 A sizable proportion of human cancer patients have
mutated tumor-suppressor genes.

Biological repair mechanisms

1.
2.
3.
4.
Living cells have evolved a series of enzymatic
systems that repair DNA damage in a variety of
ways.
Prevention of errors before they happen
Direct reversal of damage
Excision-repair pathways
Post replication repair
Prevention of errors before they
happen


Some enzymatic systems neutralize potentially
damaging compounds before they even react
with DNA.
One example of such a system is the
detoxification of superoxide radicals produced
during oxidative damage to DNA: the enzyme
superoxide dismutase catalyzes the conversion
of the superoxide radicals into hydrogen
peroxide, and the enzyme catalase, in turn,
converts the hydrogen peroxide into water.
Direct reversal of damage
Fig: Repair of a UV-induced pyrimidine photodimer by a
photoreactivating enzyme, or photolyase. The enzyme
recognizes the photodimer (here, a thymine dimer) and
binds to it. When light is present, the photolyase uses
its energy to split the dimer into the original
monomers.
Excision-repair pathways
• In E. coli, the products of the uvrA, B, and C genes
constitute the excinuclease.
•The UvrA protein, which recognizes the damaged DNA,
forms a complex with UvrB and leads the UvrB subunit
to the damage site before dissociating.
• The UvrC protein then binds to UvrB. Each of these
subunits makes an incision.
•The short DNA 12-mer is unwound and released by
another protein, helicase II.
•The human excinuclease is considerably more complex
than its bacterial counterpart and includes at least 17
proteins. However, the basic steps are the same as
those in E. coli.
Excision-repair pathways
Fig:
Schematic
representation of events
following
incision
by
UvrABC exinuclease in E.
coli. First, the UvrA
subunit recruits the UvrB
subunit to the damage site
before dissociation. Then,
as shown here, DNA
helicase II mediates the
release of a segment of
the DNA bounded by two
nicks in the same strand of
DNA. The UvrC protein is
also displaced at this point.
The subsequent repair
synthesis displaces UvrB.
Coupling of transcription and repair

In both eukaryotes and prokaryotes, there is a preferential
repair of the transcribed strand of DNA for actively
expressed genes.
Fig: Nucleotide excision repair is coupled to transcription. This model
for coupled repair in mammalian cells shows RNA polymerase (pink)
pausing when encountering a lesion. It undergoes a conformational
change, allowing the DNA strands at the lesion site to reanneal.
Protein factors aid in coupling by bringing TFIIH and other factors to
the site to carry out the incision, excision, and repair reactions. Then
transcription can continue normally.
Postreplication repair
2.
Mismatch repair
Recombinational repair
1.
Mismatch repair
1.


Some repair pathways are capable of recognizing
errors even after DNA replication has already
occurred.
One such system, termed the mismatch repair
system, can detect mismatches that occur in DNA
replication.
Mismatch repair

Works in three steps;
Recognize mismatched base pairs.
2. Determine which base in the mismatch
is the incorrect one.
3. Excise the incorrect base and carry out
repair synthesis.
1.
Mismatch repair in humans. (1) Mispairs and misaligned bases arise in the
course of replication. (2) The G–T-binding protein (GTBP) and the human
MutS homolog (hMSH2) recognize the incorrect matches. (3) Two
additional proteins, hPMS2 and hMLH1, are recruited and form a larger
repair complex. (4) The mismatch is repaired after removal, DNA
synthesis, and ligation.
Recombinational repair
Schemes for postreplication repair. (a) In recombinational repair,
replication jumps across a blocking lesion, leaving a gap in the new
strand. A recA-directed protein then fills the gap, using a piece
from the opposite parental strand (because of DNA complementarity, this filler will supply the correct bases for the gap).
Finally, the RecA protein repairs the gap in the parental strand.
Repair defects and human diseases
Table: Human Diseases with DNA-Repair Defects
The production of a wild-type phenotype when two different mutations
are combined in a diploid or a heterokaryon called complementation.
Assignment: Chromosomal aberrations
Topic 1: Changes in the number
chromosomes. Aneuploidy and euploidy.
of
Topic 2: Changes in the structure of
chromosomes,
deficiency,
duplication,
inversion and translocation.
Marks distribution:
Data collection, arrangement of data, Comprehensive description of
topic. How well described?
Group: Maximum three students in a group. Explain contribution of
each student in one line.