Download Lecture 3. MUTATIONS and DNA REPARATION A. Mutations have

Lecture 3. MUTATIONS and DNA REPARATION
A. Mutations have been reported in all organisms. The mutations cause variations in traits
and heredity diseases of human beings. Mutations occur spontaneously in a random manner by
environmental effect, or they can be induced by physical factors (X-rays, UV-radiation) or
chemicals, called mutagens.
1. Mutations are caused by changing of the DNA structure. According to their size and
quality mutations are divided to genes’ mutations and chromosomal mutations.
2. Gene’s mutations (point mutations) are caused by changes in nucleotide sequence of
DNA; chromosomal mutations are caused by changes in chromosomes structure.
3. A changes in DNA sequence are reflected in the changes of corresponding RNA or
protein:
DNA → RNA → protein
B. Types of nucleotides’ alterations in DNA.
1. Substitution – one nucleotide is replaced by another, it is one of the most common
type of the mutation. It can be divided in two subtypes:
2. Transition – one purine is replaced by another purine, or one pyrimidine is replaced
by another pyrimidine: GC - AT.
3. Transversion - one purine is replaced by a pyrimidine: A - C
G–T
II. Frame shift mutations – occur due to insertion or deletion one or more base pairs
(b.p.). Deletion or insertion of nucleotides cause the break of the message downstream, it results
in the production of an incorrect protein.
Wild type
ATG ACC AGG TCA
DNA strand
Substitution
ATG ACT AGG TCA
Insertion (A)
ATG ACAC AGG TCA
Deletion
(missing C)
ATG AC AGG TCA
III. Missense mutations
With a missense mutation, the new nucleotide alters the codon so as to produce an altered amino
acid in the protein product.
EXAMPLE: sickle-cell disease The replacement of A by T at the 17th nucleotide of the gene for
the beta chain of hemoglobin changes the codon GAG (for glutamic acid) to GTG (which
encodes valine). Thus the 6th amino acid in the chain becomes valine instead of glutamic acid.
Nonsense mutations
With a nonsense mutation, the new nucleotide changes a codon that specified an amino
acid to the STOP codon (TAA, TAG, or TGA). Therefore, translation of the messenger RNA
transcribed from this mutant gene will stop prematurely.
EXAMPLE: cystic fibrosis. The substitution of a T for a C in a gene that encodes a protein
called the cystic fibrosis transmembrane conductance regulator (CFTR) converted a glutamine
codon (CAG) to a STOP codon (TAG). The protein produced by this mutant gene had only the
first 493 amino acids of the normal chain of 1480 and could not function.
C. The molecular mechanisms of the mutations. Copy error mutations
1. Effects of the chemicals and UV-radiation
a) UV-radiation produces thymines’ dimers. They damage the DNA structure and can’t form
the H-bonds.
b) Depurination – the loss of base. It produces gaps in DNA strand. The gap may produce a
deletion or may be filled with incorrect base.
c) Deamination of DNA bases by nitrous acids – the amino group NH2 is replaced by hydroxyl
group OH.
Adenine becomes hypoxanthine (HX),
HX bonds with G, that’s why pair A -T is replaced
by G – C pair.
Cytosine becomes uracil, C-G replaced A –T.
Guanine becomes xanthin.
d) Alkylation – adding the methyl-groups. Guanine becomes adenine.
e) Tautomerization. Besides the usual molecular configuration each nitrogen base may have
some uncommon configurations of nitrogen bases are called tautomers. When nitrogen base
is in tautomeric form it cannot be linked to its complementary base: A forms a bond with C,
G – with T.
f) Bases’ analogues are unusual bases (5’-bromuracil is analogues of thymine, 2- aminopurine
is analogues of adenine);its can be incorporated into DNA chain. 5’-bromuracil (T substitute)
links with A, it has rare enol tautomeric form which links to guanine (G): adenine - 5’bromuracil (usual pair), guanine - 5’-bromuracil (unusual pair).
g) 2-aminopurine - analogues of adenine, usually pairs with T, it’s tautomer can bond with
cytosine: 2-aminopurine –T (usual pair), 2-aminopurine – C (one H-bond, unusual pair).
D. The insertion of many copies of the same triplet of nucleotides
Several genes in humans have undergone insertions of a string of 3 nucleotides repeated over
and over. A number of inherited human disorders are caused by the insertion of many copies of
the same triplet of nucleotides in such genes. Huntington's disease and the fragile X syndrome
are examples of such trinucleotide repeat diseases.
Fragile X Syndrome
A locus on the human X chromosome contains such a stretch of nucleotides in which the triplet
CGG is repeated (CGGCGGCGGCGG, etc.). The number of CGGs may be from 5 to 50
without causing a harmful phenotype (these repeated nucleotides are in a noncoding region of
the gene). Even 100 repeats usually cause no harm. However, these longer repeats have a
tendency to grow longer still from one generation to the next; to as many as 4000 repeats.
Huntington's disease
In this disorder, the repeated trinucleotide is CAG, which adds a string of glutamines (Gln) to
the encoded protein (called huntingtin). The abnormal protein increases the level of the p53
protein in brain cells causing their death by apoptosis.
Muscular Dystrophy
Some forms of muscular dystrophy that appear in adults are caused by trinucleotide (CTG)n
repeats where n may run into the thousands.
Summary of Trinucleotide-Repeat Disorders
Disorder
Huntington disease
Myotonic dystrophy
Fragile X syndrom
Trinucleotide
Repeat
CAG
CTG
CGG
Number in normal
individuals
6 - 35
5-37
6-230
Number in affected
individuals
36 – 120
37-1500
>230
Е. DNA REPARATION SYSTEMS
All organisms have special reparation systems that correct the damages of the DNA. The main
types of reparation systems are:

Photoreactivation repair system is the process whereby genetic damage caused by
ultraviolet radiation is reversed by subsequent illumination with visible or near-ultraviolet
light. Photoreactivation (light-induced repair) – correct all dimers and is activated by
visible light energy, cleaves bonds of dimers.

This repair system requires: a) visible light and b) photoreactivation enzyme (PR). PRenzyme (presents in all organisms) has RNA as cofactor.

Excision repair system (dark repair) – corrects all dimers and deamination of cytosine.
The excision repair system requires:
a) UV-specific endonucleases (Uvr A, B,C) – make a nick in the affected strand,
b) DNA-polI – synthesizes new DNA strand,
c) Ligase seals the gap between the new DNA strand and main strand.
The types of excision repair systems:

Nucleotide excision repair is used to fix DNA lesions, such as single-stranded breaks or
damaged bases, and occurs in stages. The first stage involves recognition of the damaged
region. In the second stage, two enzymatic reactions serve to remove, or excise, the
damaged sequence. The third stage involves synthesis by DNA polymerase of the excised
nucleotides using the second intact strand of DNA as a template. Lastly, DNA ligase
joins the newly synthesized segment to the existing ends of the originally damaged DNA
strand.

Base excision repair allows for the identification and removal of wrong bases, typically
attributable to deamination—the removal of an amino group (NH2)—of normal bases as
well as from chemical modification.
Other types of DNA reparation systems

Recombination repair, or post-replication repair, fixes DNA damage by a strand
exchange from the other daughter chromosome. Because it involves homologous
recombination, it is largely error free.

Mismatch repair is a multi-enzyme system that recognizes inappropriately matched bases
in DNA and replaces one of the two bases with one that "matches" the other. The major
problem here is recognizing which of the mismatched bases is incorrect and therefore
should be removed and replaced.

Adaptive/inducible repair describes several protein activities that recognize very specific
modified bases. They then transfer this modifying group from the DNA to themselves,
and, in doing so, destroy their own function. These proteins are referred to as inducible
because they tend to regulate their own synthesis.

SOS repair or inducible error-prone repair is a repair process that occurs in bacteria and
is induced, or switched on, in the presence of potentially lethal stresses, such as UV
irradiation or the inactivation of genes essential for replication. Some responses to this
type of stress include mutagenesis—the production of mutations—or cell elongation
without cell division. In this type of repair process, replication of the DNA template is
extremely inaccurate. Obviously, such a repair system must be a desperate recourse for
the cell, allowing replication past a region where the wild-type sequence has been lost.