Chapter 9: The Mutability Of DNA
I. Introduction
A. Introduction: The Fidelity Of The DNA
1. DNA is our genetic material, as it is for every organism on
earth
2. Maintaining the fidelity of the DNA is of critical importance
a. Gene expression begins with transcription of a gene,
using the non-coding strand of DNA as a template
b. Any change in DNA sequence can greatly affect the
outcome of the process of gene expression
3. A heritable change in DNA sequence is known as a mutation
a. Some mutations can have significant biological
consequences
b. Many mutations have absolutely no effect whatsoever
4. The perpetuation of the genetic material from generation to
generation depends on maintaining mutation rates at low levels
5. High rates of mutation have devastating effects depending on
the cell type
a. High rates of mutation in germ cells will result in
reduced reproductive viability and may perhaps destroy a
species
b. High rates of mutation in somatic cells may lead to death
of an organism
6. Organisms have developed methods of DNA repair to ensure
that mutations are corrected and not passed on to subsequent
generations
a. Individuals (germ line)
b. Cells
B. Introduction: 100% Fidelity Is Not Biologically Advantageous
1. Bringing the mutation rate down to zero is also not biologically
advantageous
a. All individuals would appear the same
b. The species would not be able to adjust to changing
environmental conditions
2. If the genetic material were perpetuated with perfect fidelity,
then the genetic variation necessary to drive evolution would
cease to exist and lead to extinction of life, with no new species
arising
3. Life and biodiversity depends on a balance between mutation
of DNA and the repair of DNA
4. A simpler way to think about this is that DNA repair is not
perfect, and some errors get through
C. Introduction: Sources of DNA Damage
1. DNA damage can occur due to internal or external cues
a. Internal cues generally refer to mistakes that directly
arise due to the processes DNA replication or DNA repair
b. External cues include many types of mutagens that can
cause DNA damage
2. Three important sources of mutation are as follows
a. Inaccuracies in DNA replication
b. Chemical damage to the genetic material
c. Damage due to ionizing/non-ionizing radiation
d. Transposons (not present in humans)
3. Mutations caused by errors in DNA replication, or the
movement of transposons are considered spontaneous
mutations because they occur without any extrinsic influences
4. Mutations caused by chemicals or by radiation are considered
induced mutations because they are caused by extrinsic
influences
D. Introduction: Locations of Mutations Within Genes
1. Mutations can occur within the regulatory regions of a gene or
within the protein coding region
2. Mutations within the regulatory regions generally affect the
amount of protein produced
a. Promoter regions
b. Sequences that will encode the 5’ UTR or 3’ UTR of an
mRNA
3. Mutations in the protein coding region will affect the
corresponding amino acid sequence of the peptide that will be
produced
II. Types of Mutation
A. Types of Mutation: Introduction
1. There are several different types of mutations that can occur to
the DNA
2. Some types of mutations have the opportunity to be more or
less deleterious to the organism than others
3. Types of mutations that can occur are as follows
a. Base Substitutions
b. Deletions
c. Insertions
d. Chromosomal rearrangements
B. Types of Mutations: Base Substitutions
1. Base substitutions result from a change of one nucleotide base
in the DNA sequence to another
2. Base substitutions may also be called point mutations as there
is a change in the DNA sequence at one specific point
3. With respect to the base changes anywhere in the gene, there
are two types of point mutations
a. Transitions: Base change from purine to another purine
b. Transversions: Base change from a purine to a
pyrimidine and vice versa
4. Transitions are more common than transversions
C. Types of Mutations: Mechanisms For Producing Spontaneous Base
Substitutions
1. Base substitution mutations can arise spontaneously or be
induced
2. An example of a spontaneous base substitution (transition)
occurs during a round of DNA replication in which a G is
substituted for an A
a. Purine/Purine substitution
b. Creates a mismatched G-T base pair
3. In the subsequent round of replication two products will be
created
a. One product will contain an A-T base pair at the position
where the mismatch occurred
b. One product will contain a G-C base pair where the
mismatch occurred
c. This is the point where the transition is fixed
d. Each new daughter cell will receive DNA with different
sequence
D. Types of Mutations: Base Substitutions Can Affect Regulatory Regions
or mRNA Coding Regions of Genes
1. Base substitutions can occur either in regulatory regions of
genes or in the actual coding regions of genes
2. With respect to a mutation specifically in the protein coding
region, there are three different definitions for base substitution
mutation
a. Missense mutation: The single base change results in a
change in the corresponding amino acid sequence of the
protein
b. Nonsense mutation: The single base change results in
the formation of a premature stop codon-the
corresponding protein will be truncated (also, note the
mRNA becomes unstable)
c. Silent mutation: The single base change results in no
change in the amino acid sequence of the corresponding
protein (Note: silent mutations usually occur in the third
base of a codon)
3. Base substitutions in regulatory regions can affect whether the
gene is expressed or not
E. Types of Mutations: Base Substitutions and Disease
1. Phenylketonuria is a disorder that can be caused by a base
substitution in the phenylalanine hydrolase gene
a. The mutation that leads to PKU is a transversion from a
G to a C at codon 413
b. This mutation is also a missense mutation in that it will
result in an amino acid substitution in the corresponding
protein from Pro413 to Arg 413
2. This mutation will result in a PAH enzyme that is no longer
functional, and will not be able to metabolize phenylalanine
3. This leads to a variety of developmental defects including
severe mental retardation
4. One can manage the disorder by feeding the patient a diet low
in protein, and with amino acid supplements lacking
phenylalanine
F. Types of Mutations: Insertions and Deletions
1. Insertions and deletions are generally rare as compared to
base substitutions
2. The size of insertion or deletion can be from just nucleotide to
thousands of nucleotides
a. Insertion or deletion of just one nucleotide is also called
a point mutation
b. No matter the size of insertion/deletion, it can possibly
lead to a deleterious phenotype
3. Insertions and deletions, if they occur in the protein coding
region of a gene, can affect the reading frame, which is read in
multiples of three bases (codons)
4. If the length of the insertion or deletion is not an exact
multiple of three nucleotides, then the mutation causes a
frameshift
a. The frameshift mutation will shift the phase in which the
ribosome reads the triplet codons that are 3’ to the
insertion/deletion
b. A peptide with a completely different amino acid
sequence is produced
5. Insertions and deletions of multiples of three will have no
effect on the frame
a. The codons 3’ the deletion/insertion are unaffected, and
thus, other than the insertion/deletion, the other amino
acids in the corresponding protein are unaffected
b. Other than the amino acids that are encoded by the
deleted codon(s), the rest of the amino acid sequence for
the peptide
6. Insertions/Deletions that alter frame generally have stronger
phenotypes than those that do not
G. Types of Mutations: Mechanisms Tri-Nucleotide Insertions/Deletions
1. The overall rate at which new mutations arise spontaneously
at any given site on a chromosome ranges from about 10-6 to 1011 per round of DNA replication
a. The human genome is roughly 3 x 1010 base pairs in size
b. One error per round of replication
2. Given the fact that less than 3% of the human genome is
dedicated to exons, and that the genetic code is degenerate, an
error per round of replication generally poses little problems
3. Not all of the sequence across the genome is equally
susceptible to replication error
a. Mutation prone sequences are repeats of di-, tri- or
tetranucleotide repeats
b. Known as microsatellites
c. Often implicated in human disease
4. One example of a common di-nucleotide found in the human
genome is the CA repeat
a. Replication machinery has difficulty copying these
repeats accurately due to the fact that DNA polymerases
frequently undergo slippage when copying this sequence
b. The slippage either increases or reduces the numbers of
copies of this repeat
5. Commonly seen tri-nucleotide repeats are CTG and CAG
H. Types of Mutations: Expansion of Trinucleotide Repeats Leads to
Genetic Instability
1. A select number of genes within the human genome have
trinucleotide repeats
a. The FMR1 (Fragile X Mental Retardation) gene normally
has 6-50 repeats of the sequence CGG in the sequence
encoding the 5’UTR
b. The Huntington’s gene normally has 10-26 CAG repeats
located in the coding region
2. These trinucleotide repeats can be expanded due to errors in
DNA replication
3. The trinucleotide expansion is really just a type of insertion
4. If the CAG repeats in the Huntington’s gene is expanded
beyond approximately 40 repeats can develop Huntington’s
disease
a. Neurodegenerative disorder
b. Generally has an onset after 40 years of age
c. Progressive motor disorders
d. Progressive cognitive disorders
e. Behavioral disturbances as the disease progresses
f. Eventually paralysis and death
5. The trinucleotide expansions can adopt triple helix
conformations and assume unusual DNA secondary structures
that interfere with transcription and DNA replication
III. General Classes of DNA Damage
A. General Classes of DNA Damage: Introduction
1. As we previously talked about, a mutagen is any agent that
causes an increase in the rate of mutation above the spontaneous
background
a. Chemicals
b. Radiation
c. Free Radicals
2. Damage to DNA consists of any change introducing a deviation
from the usual double-helical structure
3. There are three classes of DNA damage
a. Single base changes
b. Structural distortion
c. DNA backbone damage
B. Classes of DNA Damage: Single Base Changes
1. Single base changes affect the DNA sequence, but generally
does not have a large affect on overall DNA structure
2. One of the most common causes for a single base change is a
deamination reaction
3. Can occur spontaneously by the interaction with water or by
the action of a chemical mutagen
a. The deamination reaction replaces the amino group on
cytosine to an oxygen
b. This converts the cytosine to a uracil, leaving a G-U base
pair in the DNA
c. This G-U base pair can cause a minor distortion in the
DNA structure, but will not affect transcription or
replication
4. In vertebrates, the DNA frequently contains methylated
cytosine residues (5-methyl cytosine)
a. If these undergo a deamination reaction, then the
cytosine will be converted to a thymine
b. This will ultimately result in a change from a G-C base
pair to an A-T base pair
C. Classes of DNA Damage: Mechanisms Leading To Single Base Changes
1. Other causes of single base changes are:
a. Alkylation
b. Oxidation
c. Radiation
2. Alkylating agents such as nitrosamines lead to the formation of
O6-methylguanine
a. This base mispairs with thymine
b. This leads to a change from a G-C base pair to an A-T
base pair
3. Ionizing radiation and chemical mutagens can act as potent
oxidizing agents
a. Reactive oxygen species can generate 8-oxoguanine
(oxoG), which is a damaged guanine base containing an
extra oxygen atom.
b. OxoG can form a Hoogsteen base pair with adenine
c. Will give rise to a G-C  T-A conversion
d. This conversion is one of the most common mutations
found in cancers
D. Classes of DNA Damage: Mechanisms Leading To Structural
Distortion
1. Structural distortions are caused by agents that that can
grossly effect the structure of DNA, which may in turn inhibit
various important cellular process
a. DNA replication
b. Gene expression
2. Both chemical agents as well as radiation can cause structural
damage to occur
3. There are three classes of agents that can lead to structural
distortions
a. Ultraviolet Radiation
b. Intercalating agents
c. Base analogs
4. Absorption of light at a wavelength of 260 nm can cause
significant structural changes in the DNA
a. Can introduce pyrimidine dimers in the DNA between
neighboring thymines
b. Dimers are also termed cyclobutane-pyrimidine dimers
(CPD) because a cyclobutane ring is formed between
carbon atoms 5 and 6 in the thymine rings
c. More rarely pyrimidine neighboring cytosines and
thymines
5. The covalent bonds formed between the two thymines disrupts
the normal base pairing with adenines and distort the overall
physical structure of the DNA
a. Disrupts transcription
b. Disrupts replication
6. Due to the structural distortions, pyrimidine dimers cannot
remain within the DNA and are removed by the repair machinery
E. Classes of DNA Damage: Structural Distortion
1. Intercalating agents (chemical) are also a source that
commonly causes structural distortions in DNA
2. By definition, an intercalating agent will cause the distortion
by chemically inserting itself within the base-paired structure
3. A common intercalating agent that is used in molecular labs is
ethidium bromide
a. Contains polycyclic rings which are common amongst
intercalating agents
b. The polycyclic rings are flat and are able to insert within
the bases
c. The ethidium bromide will insert in a manner which
allows it to stack with the other base pairs causing
significant structural distortions
4. This distortion will result in either deletion or insertion
mutations in the following round of DNA replication
5. The last class of agents that cause structural distortions are the
base analogs
6. Base analogs are by definition similar in structural to the
commonly found nitrogenous bases in DNA
a. Can be taken up by normal cells
b. Can be incorporated into DNA
c. Can form base pairs, albeit inappropriately
7. 5-Bromouracil is a base analog of thymine
a. 5-bromouracil does not pair with adenine as thymine
does
b. 5-bromouracil base pairs with guanine
c. These mispairs cause structural changes to the DNA, and
result in mutation
F. Classes of DNA Damage: DNA Backbone Damage
1. DNA backbone damage results from either double strand
breaks or the formation of abasic sites from DNA (loss of a base
from the DNA)
2. DNA backbone damage can be induced by a number of
different agents
a. Ionizing radiation (X-rays and radioactive materials)
b. Action of water
3. Abasic sites are generated spontaneously by the formation of
unstable base adducts due to the action of water
4. The DNA then becomes depurinated by hydrolysis of the Nglycosyl linkage
a. Normally the sugar-purine bonds are relatively labile
b. Hydrolysis of the N-glycosyl linkage removes the purine
base and leaves a hydroxyl group in its place
5. The double strand breaks are caused by ionizing radiation
6. The ionizing radiation attacks the deoxyribose sugar by
directly or indirectly generating reactive oxygen species
7. Since the double strand breaks affect both strands of DNA, they
can be among the most severe form of DNA damage
IV. Consequences of DNA Damage
A. Although these classes of DNA damage do occur, our cells do have
mechanisms to go about fixing the damage
B. However, in the process of fixing the damage, the base sequence of
the DNA becomes changed
C. Therefore, each class of DNA damage will have its own important
consequences
1. What type of damage occurs and how the DNA sequence
becomes changed
2. Where the damage occurs
D. The enzymes involved in adding DNA sequence during the repair
process are error prone, leading to incorporation of incorrect
nucleotides
E. DNA damage has stronger effects on male fertility than female
fertility
F. DNA damage also can result in cancer formation