Download DNA MUTATIONS AND THEIR REPAIR

Lecture 2
DNA MUTATIONS AND THEIR REPAIR
AND
RECOMBINANT DNA
Objectives:
1. To understand how errors may occur during replication and their repair mechanism.
2. To define recombination. List the use of recombinant DNA techniques in medicine
DNA MUTATIONS AND THEIR REPAIR
MUTATIONS: are any permanent changes in the genetic material (usually DNA or RNA) of a cell.
Mutations can be caused by copying errors in the genetic material during cell division and by
exposure to radiation, chemicals, or viruses, or can occur deliberately under cellular control during
the processes such as meiosis or hypermutation. Mutagenesis is the process by which mutations
arise.
Types of mutations

Point mutations are usually caused by chemicals or malfunction of DNA replication and
exchange a single nucleotide for another. Most common is the transition that exchanges a
purine for a purine or a pyrimidine for a pyrimidine (A ↔ G, C ↔ T). A transition can be
caused by nitrous acid, base mispairing, or mutagenic base analogs such as 5-bromo-2deoxyuridine (BrdU). Less common is a transversion, which exchanges a purine for a
pyrimidine or a pyrimidine for a purine (C/T ↔ A/G). A point mutation can be reversed by
another point mutation, in which the nucleotide is changed back to its original state (true
reversion) or by second-site reversion (a complementary mutation elsewhere that results in
regained gene functionality). Point mutations are called silent, missense or nonsense
mutations, depending on whether the erroneous codon codes for the same amino acid
(silent), a different amino acid (missense) or a stop, which can truncate the protein
(nonsense).
o Insertions add one or more extra nucleotides into the DNA. They are usually
caused by transposable elements, or errors during replication of repeating elements
(e.g. AT repeats). Most insertions in a gene can cause a shift in the reading frame
(frameshift) or alter splicing of the mRNA, both of which can significantly alter the
gene product. Insertions can be reverted by excision of the transposable element.
o Deletions remove one or more nucleotides from the DNA. Like insertions, these
mutations can alter the reading frame of the gene. They are irreversible.
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Prof. Dr. H.D.El-Yassin
2013
Lecture 2
Original DNA molecule
ACGAGTGTGCGATCACCT
Transcription
Insertion of extra T
unit
mRNA
UGCUCACACGCUAGUGGA
Translation
Peptide
Cys Ser His Ala Ser Gly
Extra unit
Mutant DNA
ACGATGTGTGCGATCACCT
Transcription
Mutant mRNA
UGCUACACACGCUAGUGGA
Translation
Mutant peptide
Cys Tyr Thr Arg
The mutant peptide not only has the incorrect order of amino acids but also shorter.
Causes of mutation
Two classes of mutations are spontaneous mutations (naturally occurring) and induced mutations
caused by mutagens.
Spontaneous mutations on the molecular level include:
a. Errors in replication. If a base that is noncomplementory to the template base added
during replication, then a mispairing or mismatch occurs. This leads to a mutation during
the next round of replication if the error is not repaired.
b. Errors that occur during recombination events. (Recombinant DNA: molecules of DNA
formed by inserting portions of DNA from one organism into DNA of another.
c. Tautomerism
a. Keto ↔ Enol
b. Amino ↔ Imino
d. Substitutions - one base is replaced by another. If this mutation occurs within the coding
sequence of a gene, it may lead to the use of a different amino acid or generate a stop
codon. Substitutions fall into two categories:
o Transitions - one purine is replaced by another (A -> G or G -> A), or one
pyrimidine is replaced by another (C -> T or T -> C)
o Transversions - a purine is replaced by a pyrimidine (A -> C or T; G -> C or T), or a
pyrimidine is replaced by a purine (C -> A or G; T -> A or G)
e. Frameshift mutation (insertion or deletion on one strand), usually through a polymerase
error when copying repeated sequences
a. Deletions - one or more bases is removed. Unless this mutation results in the loss
of a multiple of three bases, a frame-shift will occur in coding sequences,
drastically altering every codon downstream of the mutation, and therefore the final
amino acid composition of the protein.
b. Insertions - one or more bases is added. The effects are the same as deletions,
resulting in frame-shift mutations.
f. Oxidative damage caused by oxygen radicals
g. Spontaneous changes:
a. Deamination of cytosine (C) to form uracil (U).
b. Spontaneous depurination. Purines are less stable under normal cellular
conditions than pyrimidines. The glycosidic bond that links purines to the sugarphosphate backbone of DNA often is broken. If these purines are not replaced
before a round of replication, any base may be added to complement the missing
base during replication.
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Prof. Dr. H.D.El-Yassin
2013
Lecture 2
Induced mutations on the molecular level include:
1. Chemical mutations
1. Nonalkylating agents. For example:
i.
Formaldehyde (HCHO) reacts with amine groups and cross-links DNA,
RNA and proteins.
ii.
Hydroxylamine (NH2OH) specifically reacts with cytosine to form
derivatives that pair with adenines instead of guanines. This change lead
to a transversion (in which a purine is replaced by a pyrimidine or a
pyrimidine is replaced by a purine.
iii.
Nitrous acid (HNO2) oxidatively deaminates cytosine, adenines, and
guanines to form uracil, hypoxanthines, and xanthines respectively. These
changes results in transitions (in which a purine is replaced by another
purine or one pyrimidine is replaced by another pyrimidine.
b. Alkylating agents: these act as strong electrolytes, which become linked to many
cellular nucleophiles in particular the sevenths nitrogen of the guanine in the DNA. This
linkage causes breakage of DNA.
2. Irradiation
a. Ultraviolet (UV) light (200-400 nm) induces dimerization of adjacent pyrimidines,
particularly adjacent thymines. This direct mutation of DNA distorts the DNA structure,
inhibits transcription, and disrupts replication until it is repaired.
b. Ionizing radiation, such as Roentgen rays (x rays) and gamma rays (γ-rays) can
cause extensive damage to DNA including opening purine rings, which lead to
depurination, and breaking phosphodiester bonds.
DNA repair
Some mutations in DNA can be repaired because the genetic information is stored on both strands
of DNA. The unaffected strand can be used as a template to fix the damaged strand. Chemicals,
ionizing radiation and ultraviolet light can cause breakage of the phosphodiester bonds in the DNA
backbone, and the bases themselves can be altered, lost, or covalently cross-linked.
UV light can cause adjacent pyrimidine bases to become covalently joined, forming a pyrimidine
dimer.
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Prof. Dr. H.D.El-Yassin
2013
Lecture 2
This lesion is removed by an excinuclease,
an enzyme which excises a 12 bp fragment
surrounding the dimer. DNA polymerase I
fills in the gap and DNA ligase seals the
break:
An important aspect in the repair of DNA,
especially in base mismatches, is the ability
to distinguish between strands.
Parental DNA can be distinguished from the
newly synthesized strand by methylation of
adenine residues. Specific methylases
react with adenine in GATC sequences.
This enzyme takes time to operate, so in
newly synthesized DNA the daughter strand
won't be methlyated and the repair
mechanisms can identify the parental
strand and use it as a template to correct
the unmethylated strand.
Defects in the repair mechanism of DNA
can lead to cancer.
Xeroderma pigmentosum can result from
a deficiency in the excinuclease which
removes pyrimidine dimers. Individuals with
this disease frequently die from metastases of malignant skin tumors before the age of 30.
The DNA repair process must be constantly operating, to correct rapidly any damage in the DNA
structure.
As cells age, however, the rate of DNA repair can no longer keep up with ongoing DNA damage.
The cell then suffers one of three possible fates:
1. an irreversible state of dormancy, known as senescence
2. cell suicide, also known as apoptosis or programmed cell death
3. cancer
Most cells in the body become senescent. Then, after irreparable DNA damage, apoptosis occurs.
In this case, apoptosis functions as a "last resort" mechanism to prevent a cell from becoming
cancerous and endangering the organism.
When cells become senescent, alterations in their gene regulation cause them to function less
efficiently, which inevitably causes disease. The DNA repair ability of a cell is vital to its normal
functioning and to the health and longevity of the organism.
Nuclear versus mitochondrial DNA damage
In human, and eukaryotic cells in general, DNA is found in two cellular locations:
1. inside the nucleus : (nDNA) exists in large scale aggregate structures known as
chromosomes which are composed of DNA wound up around bead-like proteins called
histones. Whenever the cell needs to access the genetic information encoded in nDNA
it will unravel the required section, read it, and then allow it to wind up once more in its
protected conformation
2. inside the mitochondria: (mtDNA) exists in single or multiple copies of a circular loop
without any histone association.
Consequently, mtDNA is far more prone to damage than nDNA because it lacks the structural
protection afforded by histone proteins. In addition, the highly oxidative environment inside
mitochondria that exists due to the constant production of adenosine triphosphate (ATP) makes
mtDNA even more prone to damage. Even though human mtDNA encodes only 13 proteins, a
malfunctioning mitochondrion can activate apoptosis.
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Prof. Dr. H.D.El-Yassin
2013
Lecture 2
The types of molecules involved and the mechanism of repair that takes place is based on:
1. the type of damage on the DNA molecule
2. whether the cell has entered into a state of senescence
3. the phase of the cell cycle that the cell is in
Single strand and double strand DNA damage
Single strand damage
To repair damage to one of the two helical domains of DNA, the other strand
must remain intact so that a replacement of damaged information can be made
by the information from the undamaged copy. There are numerous mechanisms
by which DNA repair can take place. These include
1. base excision repair (BER), which repairs damage due to alkylation or
deamination;
2. nucleotide excision repair (NER), which repairs damage by UV light; and
3. mismatch repair (MMR), which corrects errors of DNA replication and
recombination.
Cells that divide have an additional means of DNA repair via DNA polymerases.
Cells that do not divide (such as brain and heart cells) cannot use this important
DNA repair mechanism.
Double strand damage
Most cells in the body have two copies of each chromosome, which becomes
very useful during double strand damage. When damage occurs to both DNA
strands, the only way that it can be repaired is by homologous recombination using the intact
chromosome copy. This allows a damaged chromosome to be replaced, using the sister of the
chromosome pair as the template.
DNA repair rate is variable
If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors
can overwhelm the cell and result in senescence, apoptosis or cancer. Inherited diseases
associated with faulty DNA repair functioning result in premature aging (e.g. Werner's syndrome)
and increased sensitivity to carcinogens (e.g Xeroderma Pigmentosum).
Studies in smokers have found that, for people with a mutation that causes them to express less of
the powerful DNA repair gene , their vulnerability to lung and other smoking related cancers are
increased. Single nucleotide polymorphisms (SNP) associated with this mutation can be clinically
detected.
Hereditary DNA repair disorders
Defects in the NER mechanism are responsible for several genetic disorders, including:



xeroderma pigmentosum: hypersensitivity to sunlight/UV, resulting in increased skin
cancer, incidence and premature aging
Cockayne syndrome: hypersensitivity to UV and chemical agents
trichothiodystrophy: sensitive skin, brittle hair and nails
Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of
developmental neurons.
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Prof. Dr. H.D.El-Yassin
2013
Lecture 2
Other DNA repair disorders include:


Werner's syndrome: premature aging and retarded growth
Bloom's syndrome: sunlight hypersensitivity, high incidence of malignancies
Chronic DNA repair disorders
Chronic disease can be associated with increased DNA damage. For example, smoking cigarettes
causes oxidative damage to the DNA and other components of heart and lung cells, resulting in the
formation of DNA adducts (molecules that disrupt DNA). DNA damage has now been shown to be
a causative factor in diseases from atherosclerosis to Alzheimer's, where patients have a lesser
capacity for DNA repair in their brain cells. Mitochondrial DNA damage has also been implicated in
numerous disorders.
Medicine & DNA repair modulation
There is a vast body of evidence that has correlated DNA damage to death and disease. As
indicated by new overexpression studies, increasing the activity of some DNA repair enzymes
could decrease the rate of aging and disease. This may result in the development of human
interventions that can add many healthy and disease-free years to an aging population. Not all
DNA repair enzymes are beneficial when overexpressed, however. Some DNA repair enzymes
can introduce new mutations in healthy DNA. Reduced substrate specificity has been implicated in
these errors.
Cancer treatment
Procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell
to repair DNA damage and resulting in cell death. Cells that are most rapidly dividing such as
cancer cells are preferentially affected. The side effect is that other non-cancerous but similarly
rapidly dividing cells such as stem cells in the bone marrow are also affected. Modern cancer
treatments attempt to localize the DNA damage to cells and tissues only associated with cancer.
Gene repair
Unlike the multiple mechanisms of endogenous DNA repair, gene repair (or gene correction)
refers to a form of gene therapy, which precisely targets and corrects chromosomal mutations
responsible for a disorder. It does so by replacing the flawed DNA sequence with the desired
sequence, using techniques such as oligonucleotide-directed mutagenesis. Genetic mutations
requiring repair are normally inherited, but in some cases they can also be induced or acquired
(such as in cancer).
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Prof. Dr. H.D.El-Yassin
2013
Lecture 2
RECOMBINANT DNA
Recombinant DNA is the general name for taking a piece of one DNA, and combining it with
another strand of DNA. Thus, the name recombinant.
Recombinant DNA is also sometimes referred to as “chimera." By combining two or more
different strands of DNA, scientists are able to create a new strand of DNA.
The most common recombinant process involves combining the DNA of two different
organisms.
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Prof. Dr. H.D.El-Yassin
2013
Lecture 2
Nice to know:
How is Recombinant DNA made?
There are three different methods by which Recombinant DNA is made. They are:
1.
Transformation
1. The first step in transformation is to select a piece of DNA to be inserted into a vector.
2. The second step is to cut that piece of DNA with a restriction enzyme and then ligate the DNA insert
into the vector with DNA Ligase.
2. Phage Introduction
Phage introduction is the process of transfection, which is equivalent to transformation, except a phage is used instead of
bacteria. In vitro packagings of a vector is used.
3. Non-Bacterial Transformation
This is a process very similar to Transformation. The only difference between the two is non-bacterial does not use
bacteria such as E. Coli for the host. The DNA is injected directly into the nucleus of the cell being transformed.
How does rDNA work?
Recombinant DNA works when the host cell expresses protein from the recombinant genes. A
significant amount of recombinant protein will not be produced by the host unless expression
factors are added. Protein expression depends upon the gene being surrounded by a collection of
signals which provide instructions for the transcription and translation of the gene by the cell
Why is rDNA important?
• Better Crops (drought & heat resistance)
• Recombinant Vaccines (i.e. Hepatitis B)
• Prevention and cure of sickle cell anemia
• Prevention and cure of cystic fibrosis
• Production of clotting factors
• Production of insulin
• Production of recombinant pharmaceuticals
• Plants that produce their own insecticides
• Germ line and somatic gene therapy
The rapid development of techniques in the field of molecular biology is revolutionizing the practice
of medicine. The potential uses of these techniques for the diagnosis and treatment of disease are
vast.
Clinical applications:
1. Techniques of molecular biology are used in the prevention and treatment of disease.
For example, recombinant DNA techniques provide human insulin for the treatment of
diabetes, Factor VIII for the treatment of hemophilia, and vaccines for the prevention of
hepatitis. Although treatment of disease by gene therapy is in the experimental phase of
development, the possibilities are limited only by the human imagination and, of course,
by ethical considerations.
2. Techniques. To recognize normal or pathologic genetic variations, DNA must be
isolated from the appropriate source, and adequate amounts must be available for
study.
Techniques for isolating and amplifying genes and studying and manipulating
DNA sequences involve the use of:
(a) restriction enzymes,
(b) cloning vectors,
(c) polymerase chain reaction (PCR),
(d) gel electrophoresis,
(e) blotting onto nitrocellulose paper, and the preparation of
(f) labeled probes that hybridize to the appropriate target DNA sequences.
Gene therapy involves isolating normal genes and inserting them into diseased cells so that the
normal genes are expressed, permitting the diseased cells to return to a normal state. Students
must have at least a general understanding of recombinant DNA techniques to appreciate their
current use and the promise they hold for the future.
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Prof. Dr. H.D.El-Yassin
2013
Lecture 2
Conclusions:
1. Errors occurring during replication could lead to deleterious mutations. However, many
errors are corrected by enzyme activities associated with the complex at the replication
fork. The error rate is thus kept at a very low level. Damage to DNA molecules also causes
mutations. Repair mechanisms correct DNA damage, usually by removing and replacing
the damaged region. The intact, undamaged strand serves as a template for the DNA
polymerase involved in the repair process.
2. Recombination. Although cells have mechanisms to correct replication errors and to repair
DNA damage, some genetic change is desirable. It produces new proteins or variations of
proteins that may increase the survival rate of the species. Genetic change is produced by
unrepaired mutations and by a mechanism known as recombination in which portions of
chromosomes are exchanged.
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Prof. Dr. H.D.El-Yassin
2013