Lecture 5
Molecular biology
Dr. Israa Al- Yasiri 1
DNA Mutation and Repair
Mutation:
One of the properties of the genetic material, as outlined in the module on nucleic
acids, is the ability to exhibit variation over time. This property was necessary to
explain why individuals within a population are not all genetically identical, and to
explain how organisms evolve. Mutation is defined as a failure to store genetic
information faithfully. Changes in genetic information can be reflected in the
expression of that information (i.e. in the proteins produced). In other words, mutation
accounts for the variability in the genetic information. Mutation is therefore a doubleedged sword. One one hand, mutation is necessary to introduce variation into the gene
pool of a population. Genetic variation has been shown to correlate with species
fitness. On the other hand, most mutations are deleterious to the individuals in which
they occur. So mutation is good for the population, but generally not so good for the
individual.
Somatic vs. Gametic Mutations
The consequences of a mutation depend upon where in an individual they occur.
Some mutations occur in regular body cells; these are somatic mutations. For
example, someone who spends too much time suntanning might experience a
mutation in a skin cell. The consequences of such a mutation are felt only by the
individual. The skin cell may develop some problem (such as cancer, perhaps) as a
result of the mutation, but because the mutation occurred only in a skin cell, it would
not be passed on to subsequent generations. Some mutations occur in germline cells.
These cells produce the gametes; therefore, they are gametic mutations. In most
cases, such mutations wouldn't even be noticed by the individual. After all, the
gametes don't play a prominent role in the day-to-day function of the individual.
These mutations, in contrast to the somatic mutations, will be passed on to the next
generation, because they occur in the cells that produce the next generation.
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Molecular biology
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Spontaneous vs. Induced Mutations
Some mutations arise as natural errors in DNA replication (or as a result of unknown
chemical reactions); these are known as spontaneous mutations. The rates of such
mutations have been determined for many species. E. coli has a spontaneous mutation
rate of 1/108 (one error in every 108 nucleotides replicated). Humans have a higher
spontaneous mutation rate: between 1/106 and 1/105 (probably as a result of the higher
complexity of human replication).
Mutations can also be caused by agents in the environment; these are induced
mutations. Induced mutations increase the mutation rate over the spontaneous rate.
Looking at a single mutation in an individual, one cannot tell if the mutation was
spontaneous or induced. Induced mutations can only be discerned by looking at the
mutation rate in a population, and comparing it to the spontaneous mutation rate for
the species. If the observed mutation rate is higher, then induced mutations can be
assumed. Agents in the environment that cause an increase in the mutation rate are
called mutagens.
Mutations: Random and Reversible
The spontaneity of many mutations should suggest to you that the process is random.
Mutations do not occur in response to a stimulus. In other words, bacteria do not
mutate to become antibiotic resistant as a response to exposure to antibiotics. Instead,
out of all of the mutations occurring in a population of bacteria, some (a miniscule
percentage) will cause antibiotic resistance. If that antibiotic is encountered, those
bacterial cells with that particular mutation will survive; the vastmajority of the cells
that do not have the mutation will die. Mutations can be reversible. If a mutation
occurs once in a gene, there is a very small probability that the mutated base could
mutate back to its original form. Alternatively, there are occasions when a mutation in
a second, separate gene will return the phenotype of the organism to a wild type
appearance (a rare case of two wrongs making a right). This kind of mutation is
known as a supressor mutation.
Effects of Mutation : Mutations can affect individuals in a variety of ways. Among
the consequences of mutation are the following:
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Dr. Israa Al- Yasiri 3
Change in a morphological trait. This refers to an obvious change in some
physical characteristic of an organism. Most of the mutant phenotypes we
have observed in this course have been of this type (for example, short plants
instead of tall).

Nutritional or biochemical variation. A mutation may occur in a gene that
encodes an enzyme involved in a metabolic pathway, such as an enzyme
involved in the biosynthesis of an amino acid. If this occurs, the organism can
no longer synthesize the amino acid, and must obtain from dietary sources.

Change in behavior. These are hard to characterize, and there are few known
examples of specific behaviors affected by a single gene. In one example,
Drosophila mating behavior was found to be affected by a mutation. Mutant
male flies were no longer able to distinguish between males and females, and
tried to mate with any fly available!

Changes in gene regulation. If a mutation occurs in a gene encoding a
transcription factor, it could affect when and where the genes controlled by
that transcription factor are expressed. This will be addressed in the modules
on gene regulation.

Lethality. Some mutations are lethal to an organism, like the yellow coat color
allele in mice (as outlined in the module on extensions of Mendelism) or the
Huntington's allele of humans.
The Molecular Basis of Mutation:
There are two basic types of mutations:

base substitutions - this is the replacement of one base by another. For
example, if a DNA molecule usually contains guanine at a certain position, but
adenine takes the place of the guanine, then a base substitution has occurred.
There are two types of base substitutions:
o
transitions - these involve the replacement of a purine with the other
purine, or the replacement of a pyrimidine with the other pyrimidine.
o
transversions - these involve the replacement of a purine with a
pyrimidine or vice versa.
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Molecular biology
Dr. Israa Al- Yasiri 4
(Question: if a transition occurs on one strand of DNA, what type of change must
occur on the complementary strand in order to maintain complementary base
pairing?)

frameshift mutations - these change the reading frame of a gene. There are
two types of framehift mutations:
o
insertions - as the name implies, these involve the insertion of one or
more extra nucleotides into a DNA chain.
o
deletions - these result from the loss of one or more nucleotides from a
DNA chain.
(Insertion or deletion of one or two nucleotides will change the reading frame of the
genetic code. What would happen to the reading frame if three nucletides were
inserted or deleted? To further consider the effects of frameshift mutations on reading
of the genetic code, go to the genetic code module.)
Base substitutions and insertions or deletions of one nucleotide are also known as
point mutations (because they occur at a single point on a chromosome).
Causes of Mutations
Base substitutions are generally caused by changes in the way that nucleotides base
pair. One way this occurs is through tautomeric shifts. The chemical nature of the
bases of DNA is such that rare but natural, spontaneous fluctuations in the bonds of
the bases can occur. These fluctuations can briefly affect the way a base forms
hydrogen bonds. For example, adenine, when it undergoes a tautomeric shift, will
base pair with cytosine. Therefore, if a tautomeric shift occurs during replication, the
wrong nucleotide can be inserted in the newly-synthesized DNA. The bases usually
switch back to their normal form quickly, but by that time, it might be too late.
Base substitutions can also be caused by chemical modification of the bases. One type
of chemical modification is caused by alkylating agents, such as ethylmethane
sulfonate and methylmethane sulfonate. These agents donate alkyl groups (such as
methyl and ethyl groups) to bases, affecting their base pairing. For example, when
guanine is alkylated, producing 7-ethylguanine, it will base pair with thymine. Once
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Molecular biology
Dr. Israa Al- Yasiri 5
again, if this occurs during replication, the wrong nucleotide can be inserted in the
molecule being synthesized, leading to a mutation.
Frameshift mutations can be caused by intercalating agents. These are chemical
agents that insert between adjacent base pairs (like inserting between the rungs of a
ladder). The intercalation causes a conformational change in the double helix, so that
when replication occurs, the aberrant conformation causes small deletions or
insertions to occur in the newly synthesized DNA.
Radiation
Radiation is also capable of inducing mutations in DNA. Ionizing radiation, such as
gamma rays and X-rays, depending on the energy of the radiation, can create free
radicals that result in prblems ranging from point mutations to chromosome breaks.
Ultraviolet (non-ionizing) radiation can cause mutations as well. The primary effect of
UV on DNA is the creation of thymine dimers. Thymine dimers occur when two
thymines are adjacent on a strand of DNA. UV radiation can cause the formation of a
covalent bond between the two thymines, which prevents their participation in base
pairing. Thymine dimers are very deleterious to a cell - they can completely interrupt
replication, effectively causing a cell to die. As we'll see later in this module, one of
the last-chance mechanisms of repair of thymine dimers causes the insertion of
random nucleotides in place of the region containing the thymine dimer, resulting in
several base substitutions at once.
Screening for Mutagenicity
Many chemicals found in the environment (both natural and synthetic) are capable of
causing mutations. It is useful to know whether a particular substance is mutagenic
(able to cause an increase in the mutation rate), because many mutagens are also
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carcinogens (cancer causing agents). This is because cancer is generally caused by
mutations in genes that control cell division.
Chemical compounds are tested for mutagenicity using the Ames test. This test uses
auxotrophic (mutant) strains of the bacterium Salmonella typhimurium that require
medium supplemented with histidine in order to grow. The bacteria are exposed to the
compound being tested, then plated on minimal medium (with no histidine). Only
bacteria that underwent a reverse mutation, allowing them to synthesize their own
histidine, would be able to grow under these conditions. The more mutagenic a
compound is, the more likely such a reverse mutation would be. Therefore the
bacteria growing on minimal medium can be counted, and this gives a relative
measure of how mutagenic a compound is. Using this technique, a 90% correlation
has been observed between mutagenicity and carcinogenicity.
DNA Repair
Cells have developed a number of systems designed to repair DNA damage and
correct mutations. Obviously, these mechanisms are not perfectly successful, but as
we'll see, without them mutation rates would be much higher.
Repair of Thymine Dimers
Several mechanisms are available for the removal or correction of thymine dimers
from DNA. Which mechanism is used depends upon the circumstances of the cell.

Photoreactivation - It has been observed that a brief exposure to blue light
following UV exposure can reverse the effects of the UV radiation. In other
words, the blue light can cause a thymine dimer to be corrected. This is due to
the function of an enzyme called photolyase or photoreactivation enzyme
(PRE), which cleaves the covalent bonds linking the thymine dimers using the
energy from a photon of blue light. This is essentially a reversal of the reaction
that produced the thymine dimer in the first place.
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Excision Repair - This is a repair system that doesn't require light. Instead of
just breaking the bonds of the thymine dimer (as was done by photolyase), the
excision repair system removes (excises) the region surrounding the offending
nucleotides. Several proteins are involvedin this process (in prokaryotes these
are the products of the 'uvr' genes, for 'UV repair'). The steps of excision repair
in prokaryotes are as follows:
o
The distortion in the DNA (caused by the thymine dimer) is recognized
by a protein complex. A pair of endonucleases makes nicks in the
DNA strand on either side of the thymine dimer (generally the nicks
are 12 nucleotides apart).
o
The 12-nucleotide piece of DNA between the nicks is removed, and
DNA polymerase I fills in the gap left behind.
o
DNA ligase seals the final nick in the DNA.
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Molecular biology
Dr. Israa Al- Yasiri 8
Recombination Repair - Sometimes, DNA replication will begin before a
thymine dimer can be repaired by one of the other mechanisms. When the
replication machinery hits the dimer, replication stops. Occasionally, the
replication will reinitiate just beyond the dimer, leaving a gap in the DNA.
This leaves the cell with a curious predicament: if it tries to fix the dimer by
excision repair, there is no template to use for resynthesis of the DNA. How,
then, does the DNA get repaired? The answer is that the cell uses
recombination to provide a template strand for repair synthesis. Here's how:
First, the damaged region undergoes recombination with the complemenary
strand on the other DNA molecule. One strand is exchanged between the two
DNA molecules.
This essentially transfers the gap to the DNA molecule that doesn't have the
dimer.
The gap can now be filled in by DNA polymerase I, and the dimer can be
repaired by excision, since a template strand now exists.
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A more detailed examination of the actual mechanism of recombination will
be presented later in the module.

SOS Repair - If the UV exposure is sufficiently severe, the DNA damage may
overwhelm the other repair mechanisms. In such situations, DNA replication
would almost certainly halt, and the cell would die. As a last ditch effort to
save itself, a cell activates the SOS repair system. This is a complex system, in
which a whole battery of repair mechanisms are used to try to save the cell.
One of these mechanisms allows replication to proceed across damaged
templates, even though the template can't accurately be read. As a result,
random nucleotides get inserted into the newly-synthesized DNA strand. This
mechanism is therefore error-prone, and leads to mutations, which could be
deleterious. In this case, however, the alternative is death, so mutation is
preferable.
Repair of Mutations
Cells have mechanisms for minimizing the amount of mutation that takes place. As
stated previously, these are not perfect, but they do reduce greatly the frequency of
mutation. The two mechanisms we'll consider are proofreading and mismatch
repair.

Proofreading - This occurs during DNA replication. As DNA polymerase III
adds nucleotides to the growing chain, it checks each one for correct base
pairing. If the correct nucleotide has not been inserted, the polymerase uses its
3' to 5' exonuclease activity to remove the incorrect nucleotide. The
polymerase can then carry on and insert the correct nucleotide. This is very
much like a word processor: if you type in the wrong letter, you just hit the
delete key to remove it, allowing you to type in the correct letter. (For more on
3' to 5' exonuclease activity, see the module on DNA replication.) In bacteria,
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a wrong nucleotide gets inserted for every 105 nucleotides added during
replication. Proofreading corrects most of these, so that the overall error ratein
replication is one mistake for every 107 nucleotides added.

Mismatch Repair - This mechanism is used soon after replication, to correct
errors that escaped proofreading. Because mismatched bases don't hydrogen
bond, they create a distortion in the double helix, which can be recognized and
repaired by excision repair. The question in this case is how does the repair
system recognize which strand to repair? There are two nucleotides (one on
each DNA strand) that won't base pair - which one is the wrong nucleotide?
The answer comes from DNA methylation. DNA under normal circumstances
is methylated; these methyl groups do not interfere with the function of the
DNA in any way. Newly replicated DNA is not methylated however; the
methyl groups are added enzymatically after replication. If mismatch repair is
done immediately after replication (before methylation occurs), the original
DNA strand will be methylated, and the newly-synthesized strand (the one
containing the error) will be unmethylated. The mismatch repair system
therefore repairs the unmethylated strand.
Excision repair can be used to correct other problems as well. For example, if a
deoxynucleotide containing uracil is ever inserted into a DNA molecule, the base is
detected and removed by an enzyme called uracil DNA glycosylase. This enzyme
removes the base, but leaves the sugar and phosphate in the DNA molecule. This
base-less site is then recognized by specific endonucleases, which initiate excision
repair.