Chapter 9: The Mutability Of DNA

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

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

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

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

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

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

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

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

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 

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

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

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)

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

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 10-11 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

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 10-26 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 10-29 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

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

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

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

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

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

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

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

Classes of DNA Damage: Structural Distortion

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

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

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