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Lesson 8. MUTATIONS
Module 2. Fundamental biological principles
Lesson 8
MUTATIONS
8.1 Definition of MutationMUTATIONS
A mutation is defined as an abrupt qualitative or quantitative permanent change in the DNA sequence of an organism and is one of the major causes of evolution.
Gene mutations occur in two ways viz. either they are inherited from a parent (passed from parent to child) designated as ‘Hereditary mutations’ or acquired some time during a person’s life due to replication error or environmental agents. These are rare events because of the very low mutation rates (one out of a million).
8.2 Why are Mutations Important?
Mutations have considerable biological significance and play an important role in biological diversity. The study of mutations is important because of the following reasons.
i) They may have deleterious or advantageous (rarely) consequences to an organism.
ii) They are important to geneticists for studying metabolic pathways by making variants (mutants) lacking the ability to perform a process under investigation.
iii) Mutations are important for evolutionary change as the major source of genetic variations.
iv) They can also be used as tools for mapping the location of the genes in the host’s genome.
ii) They are important to geneticists for studying metabolic pathways by making variants (mutants) lacking the ability to perform a process under investigation.
iii) Mutations are important for evolutionary change as the major source of genetic variations.
iv) They can also be used as tools for mapping the location of the genes in the host’s genome.
8.3 Classification
Mutations can be classified based on their effect on phenotype and the kind of alterations they make in the DNA sequence (genotype).
8.3.1 Effects on phenotype
Based on their effect on phenotype, they are classified as
a) Lethal mutations
b) Subvital Mutations
c) Supervital Mutations
The mutations which result in the death of the cells are called as lethal mutations.
b) Subvital Mutations
The mutations which reduce the chances of survival are called as subvital mutations.
c) Supervital Mutations
The mutations which result in the improvement of biological fitness under certain conditions are called as supervital mutations.
8.3.2 Kinds of alterations in DNA
Based on their effect on alteration in their DNA, mutations are classified as
Chromosomal Mutations (Intergenic mutations): These are the mutations which occur in different genes.
Point Mutations (Intragenic mutations): These are the mutations which occur within the same gene and affect the same. These include base pair substitutions and frame shift mutations.
i) Base Pair Substitutions – Base pair mutations result from single base substitution leading to either transitions, transversions or both. e.g.
Wild Type : GCT GAT CTT GAT CAT
Mutant : GCT GAT CGT GAT CAT
Mutations brought about by base pair substitutions are of the following types. Mutant : GCT GAT CGT GAT CAT
a) Transitions: Transitions are known to occur when there is replacement of one base with a different base of the same chemical category e.g. purine to purine or pyrimidine to pyrimidine.
ACTGCTA
ACTACTA
b) Transversions: Transversions occur when there is replacement of one base with a different base of other chemical category e.g. purine to pyrimidine or pyrimidine to purine.ACTGCTA
ACTTCTA
c) Inversions: Inversions occur when a segment of DNA is removed and reinserted in a reverse direction.CGT GCT TGC GCT CGT
CGT GCT CGT GCT CGT
ii) Frame Shift Mutations – Frame shift mutations cause either deletions or insertions of one or more nucleotides leading to shift in the reading frame. Such mutations cause changes in all the amino-acids down stream of the place of mutation. CGT GCT CGT GCT CGT
5’-ACG UAU GCG CUA GCG -3’
5’ ACG CUA UGC GCU AGC G 3’
5’-ACG UAU GCG CUA GCG -3’
5’-AGU AUG CGC UAG CG -3’
Based on qualitative effect on gene product, mutations can be classified as:
8.3.3.1 Mis-sense mutations
Mis-sense mutation is a point mutation that results from substitution of one nucleotide leading to change in codon and the proteins that differ only in single amino-acid. e. g. UUU codes for Phenyl alanine. When UUU is changed to UGU by substitution of second U with G, it codes for cysteine. Protein may or may not have normal biological activity or may have partial loss of function. An example of such mutations is sickle cell anemia wherein sixth amino-acid of hemoglobin beta chain becomes valine instead of glutamic acid.
8.3.3.2 Non-sense mutations
Non sense mutations occur due to change of codon to one of the three stop / termination codons (UGA, UAG, UAA) resulting into premature termination and partial or complete loss of function e.g. cystic fibrosis.
8.3.3.3 Silent mutations
A change in nucleotide does not cause any change in amino-acid sequence and hence does not lead to any phenotypic change. As genetic code is degenerate, most amino-acids are encoded by several different codons. Such mutations may not be able to modify the function of protein and as such can not be detected without sequencing of the gene.
8.3.3.4 Mutations in termination codons
These mutations are just reverse of the non-sense mutations. In this type of mutations, termination codon is changed to sense codon which may then produce longer polypeptides by incorporating specific amino-acids in the protein.
8.3.3.5 Mutations in non-coding sequences
Mutations which occur in introns, promoters, regulatory sequences and origin of replication etc. fall in this category. These may or may not influence the function of a gene.
8.3.3.6 Reverse mutations
It is also known as back mutation or true reversion and changes the mutation back to wild type. Such mutations lead to restoring the function of protein.
Suppressor mutations occur at sites away from the original mutation and mask or compensate for the initial mutation without reversing it.
8.3.4 Effect of mutational events
On the basis of mutational events that take place in an organism, mutations can be classified as
8.3.4.1 Spontaneous mutations
Spontaneous mutations are the ones which occur without any known cause/agent and are random. These mutations arise due to errors in replication, replication slippage, mismatches, instability of bases, oxidative damage, tautomeric shift and deamination of bases. The mutations that occur due to an error in replication or as a result of mismatches are shown in Fig. 8.1.
The DNA polymerase may incorporate a wrong base during synthesis and if proof reading activity of polymerase has not removed the wrong base before synthesis, then mutation occurs as has been shown in Fig. 8.2.
8.3.4.2 Induced mutations
Induced mutations are the ones which are induced by mutagenic agents and include physical and chemical agents. These mutagens increase the frequency of mutations.
8.3.4.2.1 Physical agents
Physical agents are of two types.
i) Ionizing Radiations (X-rays, cosmic rays, gamma rays etc.) – Ionizing radiations can randomly cause damage to all the cellular components by direct or indirect interaction. The reactive oxygen species formed by radiations can also cause damage to DNA and lead to several mutagenic and carcinogenic effects. The major effect is due to strand breaks. These radiations are widely used in tumor therapy.
ii) Non-ionizing Radiations (UV rays) - These are low energy radiations and hence do not cause any ionization. UV rays cause formation of pyrimidine dimers and most common are thymine dimmers (Fig. 8.3) as well as thymine cytidine dimers. Adjacent pyrimidines are covalently linked by the formation of a four membered ring due to saturation of 5, 6 double bonds. Pyrimidine dimers may also occur in adjacent strands and lead to distortions.
ii) Non-ionizing Radiations (UV rays) - These are low energy radiations and hence do not cause any ionization. UV rays cause formation of pyrimidine dimers and most common are thymine dimmers (Fig. 8.3) as well as thymine cytidine dimers. Adjacent pyrimidines are covalently linked by the formation of a four membered ring due to saturation of 5, 6 double bonds. Pyrimidine dimers may also occur in adjacent strands and lead to distortions.
8.3.4.2.2 Chemical agents
A number of chemical agents which are frequently used for studying or producing mutations are listed below.
a. Base analogues
The structure of base analogues are similar to the nitrogenous bases and hence are incorporated in DNA during its replication. The examples are 5 – bromouracil and 2 – aminopurine (Fig. 8.4). Base analogues mainly cause transitions. When 5 – bromouracil is incorporated in DNA, it pairs with adenine. It can spontaneously shift into another isomer which then pairs with guanine. On the other hand, 2-amino-purine pairs with thymine since it is an analogue of adenine. It also spontaneously shifts into another isomer which can then pair with cytosine.
b. Alkylating agents
The alkylating agents cause alkylation at the N7 position of guanine and the N3 of adenine by adding either methyl or ethyl groups. These agents act both on replicating and non-replicating DNA. The examples are mustard gas i.e. Di (2-chloroethyl) sulphide; Ethyl methane sulfonate, EMS; Ethyl ethane sulfonate, EES and N-methyl-N’-nitro-N-nirtosoguanidine (NTG). They cause both transitions and transversions. The action of EMS has been shown in .
c. Deaminating agents
These agents cause deamination of bases and do not require replication. The examples are nitrous acid and hydroxylamine. They cause transitions.
d. Intercalating dyes
Intercalating dyes intercalate (insert) between base pairs in a double helix or between ring-stacked bases in a polynucleotide chain thus distorting the structure of DNA. They can cause insertions as well as deletions, thus, leading to frame shift mutations (Fig.8.6). The examples of intercalating dyes are acridine orange, ethidium bromide and proflavin etc. (Fig. 8.7).
8.4 DNA Repair and Its Importance
DNA is the repository of hereditary information and nearly all the DNA damage is harmful. Therefore, it is essential to reduce this damage to a minimum tolerable level. There are a number of repair mechanisms which operate in prokaryotes and eukaryotes.
8.4.1 Direct reversal of damage or photoreactivation
This repair system acts on exposure to light and is mainly responsible for repair of thymine thymine dimers. It was discovered by Albert Kelner in 1949. UV damage is reversed if exposed to light. The enzyme photolyase encoded by phrA and phrB genes of E. coli is responsible for repair of the damage. The best studied example is that of Cyclobutane pyrimidine dimer (CPD) photolyase. This enzyme binds to pyrimidine dimers and use energy from visible light to split the dimers apart. This enzyme contains two chromophores i.e. Flavin adenine dinucleotide (FADH-) and the other is either methenyl-tetrahydrofolate (MTHF) or 8-hydroxy-5-deazaflavin (8-HDF). The latter gathers the light and transfer energy to FADH which then splits the dimer. The detailed mechanism has been illustrated in Fig. 8.8.
8.4.2 Dark repair or light independent systems
The following three mechanisms act independent of light and repair several types of damage that occur due to mutagenic agents.
i) Excision of damaged region, followed by precise replacement
Base excision repair, Nucleotide excision repair, Mismatch repair
Base excision repair (BER)
It is defined as a process of DNA repair wherein the altered base is excised by DNA glycosylase followed by excision of the resulting sugar phosphate. The gap is then filled in by the DNA polymerase and ligase.
Nucleotide excision repair (NER)
Nucleotide excision repair is the process wherein UV induced pyrimidine dimers or other unwanted mutations are removed by excision with uvrABC exinuclease. The gap is then filled in with the DNA polymerase.
Mismatch repair
Mismatch repair system corrects errors in DNA. The mismatch correction enzyme detects the wrong bases and removes the segment of the DNA strand containing the mismatched bases. The gap is then filled by DNA polymerase.
ii) Tolerance of DNA damage
Replicative bypass with gap formation/Recombinational repair - using other duplexes for repair.
When the template DNA strand is not available for repair such as when a replication fork meets a lesion (thymine dimer) and separates the strands before excision repair can take place, then the information lost at that site of damage is recovered by taking a corresponding segment from a separate but identical DNA molecule is called recombinational repair.
iii) SOS error-prone ‘repair’
When the cell is severely damaged, cell engages in SOS repair in order to salvage a functioning set of genetic information. It is the last resort for the cell to survive even by incorporating a wrong base.
Further Reading
Books
Fundamental Bacterial Genetics, Nancy Trun, Janine Trempy (Eds), Wiley-Blackwell, 2003, ISBN: 978-0-632-04448-1
From Genes to Genomes: Concepts and Applications of DNA Technology, 3rd Edition, Jeremy W. Dale, Malcolm von Schantz, Nicholas Plant (Eds), Wiley-Blackwell, 2011, ISBN: 978-0-470-68386-6
Microbial Genetics, 2nd Edition, Stanly R Maloy, John Cronan, David Freifelder, Narosa, ISBN: 8173196974
Molecular Biology of the Gene, Sixth Edition, James D. Watson (Editor) Cold Spring Harbour Press and Benjamin Cummings, ISBN 978-080539592-1
Internet Resources
http://en.wikipedia.org/wiki/Mutation
http://saturn.roswellpark.org/cmb/huberman/DNA_Repair/DNA_Repair.htm
Last modified: Friday, 21 September 2012, 11:24 AM