1. Chapter 9: The Mutability Of DNA

2. Introduction: The Fidelity Of The DNA
DNA is our genetic material, as it is for every organism on earth
Maintaining the fidelity of the DNA is of critical importance
Gene expression begins with transcription of a gene, using the non-coding strand of DNA as a template
Any change in DNA sequence can greatly affect the outcome of the process of gene expression
A heritable change in DNA sequence is known as a mutation
Some mutations can have significant biological consequences
Many mutations have absolutely no effect whatsoever

3. Introduction: The Fidelity of The DNA
The perpetuation of the genetic material from generation to generation depends on maintaining mutation rates at low levels
High rates of mutation have devastating effects depending on the cell type
High rates of mutation in germ cells will result in reduced reproductive viability and may perhaps destroy a species
High rates of mutation in somatic cells may lead to death of an organism
Organisms have developed methods of DNA repair to ensure that mutations are corrected and not passed on to subsequent generations
Individuals (germ line)
Cells

4. Introduction: 100% Fidelity Is Not Biologically Advantageous
Bringing the mutation rate down to zero is also not biologically advantageous
All individuals would appear the same
The species would not be able to adjust to changing environmental conditions
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
Life and biodiversity depends on a balance between mutation of DNA and the repair of DNA
A simpler way to think about this is that DNA repair is not perfect, and some errors get through

5. Introduction: Sources of DNA Damage
DNA damage can occur due to internal or external cues
Internal cues generally refer to mistakes that directly arise due to the processes DNA replication or DNA repair
External cues include many types of mutagens that can cause DNA damage
Three important sources of mutation are as follows
Inaccuracies in DNA replication
Chemical damage to the genetic material
Damage due to ionizing/non-ionizing radiation
Transposons (not present in humans)
Mutations caused by errors in DNA replication, or the movement of transposons are considered spontaneous mutations because they occur without any extrinsic influences
Mutations caused by chemicals or by radiation are considered induced mutations because they are caused by extrinsic influences

6. Introduction: Locations of Mutations Within Genes
Mutations can occur within the regulatory regions of a gene or within the protein coding region
Mutations within the regulatory regions generally affect the amount of protein produced
Promoter regions
Sequences that will encode the 5’ UTR or 3’ UTR of an mRNA
Mutations in the protein coding region will affect the corresponding amino acid sequence of the peptide that will be produced

7. Types of Mutation: Introduction
There are several different types of mutations that can occur to the DNA
Some types of mutations have the opportunity to be more or less deleterious to the organism than others
Types of mutations that can occur are as follows
Base Substitutions
Deletions
Insertions
Chromosomal rearrangements

8. Types of Mutations: Base Substitutions
Base substitutions result from a change of one nucleotide base in the DNA sequence to another
Base substitutions may also be called point mutations as there is a change in the DNA sequence at one specific point
With respect to the base changes anywhere in the gene, there are two types of point mutations
Transitions: Base change from purine to another purine
Transversions: Base change from a purine to a pyrimidine and vice versa
Transitions are more common than transversions

9. Types of Mutations: Mechanisms For Producing Spontaneous Base Substitutions
Base substitution mutations can arise spontaneously or be induced
An example of a spontaneous base substitution (transition) occurs during a round of DNA replication in which a G is substituted for an A
Purine/Purine substitution
Creates a mismatched G-T base pair
In the subsequent round of replication two products will be created
One product will contain an A-T base pair at the position where the mismatch occurred
One product will contain a G-C base pair where the mismatch occurred
This is the point where the transition is fixed
Each new daughter cell will receive DNA with different sequence

10. Types of Mutations: Base Substitutions
Base substitutions can occur either in regulatory regions of genes or in the actual coding regions of genes
With respect to a mutation specifically in the protein coding region, there are three different definitions for base substitution mutation
Missense mutation: The single base change results in a change in the corresponding amino acid sequence of the protein
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)
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)
Base substitutions in regulatory regions can affect whether the gene is expressed or not 

11. Types of Mutations: Base Substitutions
Phenylketonuria is a disorder that can be caused by a base substitution in the phenylalanine hydrolase gene
The mutation that leads to PKU is a transversion from a G to a C at codon 413
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
This mutation will result in a PAH enzyme that is no longer functional, and will not be able to metabolize phenylalanine
This leads to a variety of developmental defects including severe mental retardation
One can manage the disorder by feeding the patient a diet low in protein, and with amino acid supplements lacking phenylalanine

12. Types of Mutations: Base Substitutions and Evolution
Recently, scientists interested in Molecular Evolution have uncovered a big piece in the puzzle of the development of speech
The Fox2P gene has been implicated in this process
Fox2P encodes a regulatory transcription factor with yet unresolved targets (genes whose transcription is regulated by Fox2P)
Within the gene, there is a single base change
GA transition within the coding region
Missense mutation resulting in a change from an arginine to a histidine at codon 553
This mutation is also seen in a single human family with a history of loss of speech-more specifically, they lack the fine facial movements to produce speech
This is an example of a mutation resulting in an evolutionary advantage

13. Types of Mutations: Insertions and Deletions
Insertions and deletions are generally rare as compared to base substitutions
The size of insertion or deletion can be from just nucleotide to thousands of nucleotides
Insertion or deletion of just one nucleotide is also called a point mutation
No matter the size of insertion/deletion, it can possibly lead to a deleterious phenotype
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)

14. Types of Mutations: Insertions and Deletions
If the length of the insertion or deletion is not an exact multiple of three nucleotides, then the mutation causes a frameshift
The frameshift mutation will shift the phase in which the ribosome reads the triplet codons that are 3’ to the insertion/deletion
A peptide with a completely different amino acid sequence is produced
Insertions and deletions of multiples of three will have no effect on the frame
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
Other than the amino acids that are encoded by the deleted codon(s), the rest of the amino acid sequence for the peptide
Insertions/Deletions that alter frame generally have stronger phenotypes than those that do not

15. Types of Mutations: Mechanisms Tri-Nucleotide Insertions/Deletions
The overall rate at which new mutations arise spontaneously at any given site on a chromosome ranges from about 10-6 to per round of DNA replication
The human genome is roughly 3 x 1010 base pairs in size
One error per round of replication
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
Not all of the sequence across the genome is equally susceptible to replication error
Mutation prone sequences are repeats of di-, tri- or tetranucleotide repeats
Known as microsatellites
Often implicated in human disease
One example of a common di-nucleotide found in the human genome is the CA repeat
Replication machinery has difficulty copying these repeats accurately due to the fact that DNA polymerases frequently undergo slippage when copying this sequence
The slippage either increases or reduces the numbers of copies of this repeat
Commonly seen tri-nucleotide repeats are CTG and CAG

16. Types of Mutations: Expansion of Trinucleotide Repeats Leads to Genetic Instability
More than just the DMPK gene within the human genome has trinucleotide repeats
The FMR1 (Fragile X Mental Retardation) gene normally has 6-50 repeats of the sequence CGG in the sequence encoding the 5’UTR
The Huntington’s gene normally has CAG repeats located in the coding region
Like Myotonic Dystrophy, these trinucleotide repeats can be expanded also due to errors in DNA replication
For these diseases, the trinucleotide expansion is again really just a type of insertion
Normally a healthy individual will have between repeats
If the CAG repeats in the Huntington’s gene is expanded beyond approximately 40 repeats can develop Huntington’s disease
Neurodegenerative disorder
Generally has an onset after 40 years of age
Progressive motor disorders
Progressive cognitive disorders
Behavioral disturbances as the disease progresses
Eventually paralysis and death
The trinucleotide expansions can adopt triple helix conformations and assume unusual DNA secondary structures that interfere with transcription and DNA replication

17. General Classes of DNA Damage: Introduction
When we talk about DNA damage, we are going to be studying the interactions of mutagens and the DNA-
More specifically, the types of damage caused by each type of mutagen
How each type of damage is caused
As we previously talked about, a mutagen is any agent that causes an increase in the rate of mutation above the spontaneous background
Chemicals
Radiation
Free Radicals
Damage to DNA consists of any change introducing a deviation from the usual double-helical structure
There are three classes of DNA damage
Single base changes
Structural distortion
DNA backbone damage

18. Classes of DNA Damage: Mechanisms Leading To Single Base Changes
Single base changes affect the DNA sequence, but generally do not have a large affect on overall DNA structure
One of the most common causes for a single base change is a deamination reaction
Can occur spontaneously by the interaction with water or by the action of a chemical mutagen
The deamination reaction replaces the amino group on cytosine to an oxygen
This converts the cytosine to a uracil, leaving a G-U base pair in the DNA
This G-U base pair can cause a minor distortion in the DNA structure, but will not affect transcription or replication
In vertebrates, the DNA frequently contains methylated cytosine residues (5-methyl cytosine)
If these undergo a deamination reaction, then the cytosine will be converted to a thymine
This will ultimately result in a change from a G-C base pair to an A-T base pair

19. Classes of DNA Damage: Mechanisms Leading To Single Base Changes
Other causes of single base changes are:
Alkylation
Oxidation
Radiation
Alkylating agents such as nitrosamines lead to the formation of O6-methylguanine
This base mispairs with thymine
This leads to a change from a G-C base pair to an A-T base pair
Ionizing radiation and chemical mutagens can act as potent oxidizing agents
Reactive oxygen species can generate 8-oxoguanine (oxoG), which is a damaged guanine base containing an extra oxygen atom.
OxoG can form a Hoogsteen base pair with adenine
Will give rise to a G-C  T-A conversion
This conversion is one of the most common mutations found in cancers

20. Classes of DNA Damage: Mechanisms Leading To Structural Distortion
Structural distortions are caused by agents that that can grossly effect the structure of DNA, which may in turn inhibit various important cellular process
DNA replication
Gene expression
Both chemical agents as well as radiation can cause structural damage to occur
There are three classes of agents that can lead to structural distortions
Ultraviolet Radiation
Intercalating agents
Base analogs

21. Classes of DNA Damage: Mechanisms Leading To Structural Distortion
The nitrogenous bases in DNA optimally at a wavelength 260 which falls within the UV spectrum
Absorption of light at a wavelength of 260 nm can cause significant structural changes in the DNA
Can introduce pyrimidine dimers in the DNA between neighboring thymines
Dimers are also termed cyclobutane-pyrimidine dimers (CPD) because a cyclobutane ring is formed between carbon atoms 5 and 6 in the thymine rings
More rarely pyrimidine neighboring cytosines and thymines
The covalent bonds formed between the two thymines disrupts the normal base pairing with adenines and distort the overall physical structure of the DNA
Disrupts transcription
Disrupts replication
Due to the structural distortions, pyrimidine dimers cannot remain within the DNA and are removed by the repair machinery

22. Classes of DNA Damage: Structural Distortion
Intercalating agents (chemical) are also a source that commonly causes structural distortions in DNA
By definition, an intercalating agent will cause the distortion by chemically inserting itself within the base-paired structure
A common intercalating agent that is used in molecular labs is ethidium bromide
Contains polycyclic rings which are common amongst intercalating agents
The polycyclic rings are flat and are able to insert within the bases
The ethidium bromide will insert in a manner which allows it to stack with the other base pairs causing significant structural distortions
This distortion will result in either deletion or insertion mutations in the following round of DNA replication

23. Classes of DNA Damage: Structural Distortion
The last class of agents that cause structural distortions are the base analogs
Base analogs are by definition similar in structural to the commonly found nitrogenous bases in DNA
Can be taken up by normal cells
Can be incorporated into DNA
Can form base pairs, albeit inappropriately
5-Bromouracil is a base analog of thymine
5-bromouracil does not pair with adenine as thymine does
5-bromouracil base pairs with guanine
These mispairs cause structural changes to the DNA, and result in mutation

24. Classes of DNA Damage: Structural Distortion

25. Classes of DNA Damage: DNA Backbone Damage
DNA backbone damage results from either double strand breaks or the formation of abasic sites from DNA (loss of a base from the DNA)
DNA backbone damage can be induced by a number of different agents
Ionizing radiation (X-rays and radioactive materials)
Action of water
Abasic sites are generated spontaneously by the formation of unstable base adducts due to the action of water
The DNA then becomes depurinated by hydrolysis of the N-glycosyl linkage
Normally the sugar-purine bonds are relatively labile
Hydrolysis of the N-glycosyl linkage removes the purine base and leaves a hydroxyl group in its place

26. Classes of DNA Damage: DNA Backbone Damage
The double strand breaks are caused by ionizing radiation
The ionizing radiation attacks the deoxyribose sugar by directly or indirectly generating reactive oxygen species
Since the double strand breaks affect both strands of DNA, they can be among the most severe form of DNA damage

27. Consequences of DNA Damage: Introduction
Although these classes of DNA damage do occur, our cells do have mechanisms to go about fixing the damage
However, in the process of fixing the damage, the base sequence of the DNA becomes changed
Therefore, each class of DNA damage will have its own important consequences
What type of damage occurs and how the DNA sequence becomes changed
Where the damage occurs
The enzymes involved in adding DNA sequence during the repair process are error prone, leading to incorporation of incorrect nucleotides
DNA damage has stronger effects on male fertility than female fertility
DNA damage also can result in cancer formation