11 Mutations and unusual bases

Prof. Sunil Kumar Khare

  1. Objectives
  •  What are mutations
  •  Causes of mutations
  •  Types of mutations
  •  Effects of mutations
  1. Concept Map
  1. Description

3.1 What are mutations

 

Basis of mutations

 

DNA is a dynamic molecule which carries the primary genetic information, hence any changes occurring in it can lead to drastic consequences. The term ‘mutation’ was coined to describe any changes occurring in the DNA which might lead to alteration of the nucleotide sequence conferring major changes at the higher protein level. Mutations have both advantages and disadvantages. Mutations pave the way for the evolutionary process which lead to survival strategies in the living beings. On the contrary, high rates of mutations occurring in the somatic or the germ cells can destroy the individual or the species.

 

A well known example of mutation is cancer where the specialized cells lose the ability t o divide in a controllable manner. Therefore the cells biological system should be such where the mutations are identified and repaired by the cell’s repair system. In this module we will discuss and understand the causes of mutation, their effects and their subsequent repercussions on the individuals. The molecular basis of mutations comprise the various gene mutations arising due to several factors which facilitate their characterization. Mutations are often categorised on the basis of cell in which they originate: somatic mutations and germ-line mutations. Somatic mutations take place in non-reproductive cells. Most somatic mutations do not affect the organism or stay recessive due to the cell’s repair system. However, certain other mutations can occur at high rate and can affect the normal functioning of the organism. Germ-line mutations are the ones which occur in gametes or in cells that eventually produce gametes. Hence germ-line mutations are passed onto the future progeny, whether in the somatic cells or germ-line cells.

 

3.2. Gene mutations

  1. Base substitutions : These are the simplest DNA mutations where one base switches to another. These type of mutations are subdivided into two categories (a) transversions (b) transitions. The replacement of a base of one category by a base of the other (pyrimidine replaced by purine: C to A, C to G, T to A, T to G; purine replaced by pyrimidine: A to C, A to T, G to C, G to T) are known as transversion type of mutations whereas transitions describe the substitutions of pyrimidine with pyrimidine and purine with a purine.
  2. Base additions or deletions : This type refers to base additions or deletions. The simplest of these type occur within a single base. Hence these are further categorized into:

    a) Missense mutation– The codon for one amino acid is changed into a codon for a different amino acid

b) Nonsense mutation: The codon for one amino acid is changed into a termination codon

c) Silent mutation: The codon for one amino acid is changed into the codon for the same amino acid

 

Out of the above stated mutations, the ones which have a drastic effect on a protein structure and subsequent functions are the missense and non-sense mutation. A classic example of a missense mutation is the substitution of glutamic acid at position 6 of the β globin gene to valine. This alteration results in the formation of a defected protein, haemoglobin and hence its oxygen carrying capacity is affected. This ultimately result in modifying the shape of erythrocytes and lead to sickle cell anaemia. This condition is particularly endemic to the African population where every 1 in 500 individuals get affected.

3.3  Spontaneous and Induced type of mutations

 

Depending upon the cause of occurrence of mutations, these can be divided into:

 

(a) Spontaneous mutations: These are the mutations which result due to the occurrence of mutable events over a period of time under natural environmental conditions. They account for the major source of genetic variation in the population. The major factors responsible for spontaneous mutations are:

  1. Errors during DNA replication
  2. Due to compounds of unknown mutagenic potential
  3. Hydrolytic damage

(b) Induced mutations: The mutations which arise in an organism due to deliberate repeated exposure to a known mutagen (chemicals or ionizing radiations) are of the induced type. This type can also be artificially introduced in an experimental organism during mutagenesis studies

 

Spontaneous Mutations

  1. Hydrolytic damage- DNA undergoes damage by the action of water. The various types of hydrolytic reactions of DNA are as follows:

a) Deamination: This is the most common type of hydrolytic damage. Cytosine readily undergoes deamination to yield uracil in DNA. This uracil preferentially pairs up with adenine, thus altering the base pairing potential. Likewise, adenine deaminates to hypoxanthine while base pairing with cytosine, guanine gets converted to xanthine which bonds with cytosine with only two hydrogen bonds. This is the reason why the sites which have 5-methyl cytosine present are considered to be the hot spots of mutations (hot spots are those sites which are most prone to mutations)

b) Depurination: This occurs due to hydrolysis of glycosyl linkage which results into an abasic site (nucleotide without a base). The apurinic sites generated are unnatural and thus are responsible for DNA damage. An example of this type of mutation occurs with Aflatoxin which is a fungal toxin and causes depurination. Studies done earlier have revealed the repair system leads to the preferential insertion of an adenine at the apurinic site. This results in G·C → T·A transitions.

  1. Oxidative damage The reactive oxygen species (ROS) also tend to attack DNA as well its precursors and leave it mutated. These ROS such as superoxide radicals (O2D), hydrogen peroxide (H2O2), and hydroxyl radicals (OHD), are often generated by chemical agents, ionizing radiations or even by aerobic metabolism. For example, the oxidation of guanine leads to the generation of an oxoadduct which causes G.C→ T·A transition.

Replication errors: All types of bases in the DNA exist in tautomeric forms. Each base has two tautomeric forms which are in equilibrium with each other. The keto forms of each base are the natural occurring forms whereas the enol forms are rarely seen. A tautomeric shift result in the mispairing between bases. For example the enol form of guanine will pair with thymine whereas the imino form of cytosine pairs with adenine leading to mutations during replication.

Fig.3 Diagram showing the consequence of tautomeric shifts in the bases of DNA (a) A guanine base in one of the parental strand undergoes tautomeric shift to its enol form and pairs up with thymine (b) The other parental strand undergoes normal pairing. In the next replication, the guanine reverts to its natural keto form and pairs normally with cytosine and produce the wild type whereas the thymine leads to the addition of adenine in the opposite strand causing G.C → T·A mutations (d).

 

Induced mutations

 

a) DNA alkylation: DNA is subjected to both exogenous and endogenous alkylation. The endogenous alkylating agents are basically metabolic intermediates whereas the exogenous are contributed from air, water, food or from the anticancer drugs. It has been studied that all the alkylating agents form adducts at O- and N-atoms in nucleobases, as well as on O-atoms in phosphodiesters. The consequence of alkylation is majorly the mispairing with thymine which results in G.C → A.T transitions. Alkylating agents are therefore mutagenic and genotoxic.

 

b) Endogenous alkylating agents: As per the studies conducted, it is said that the nitrosation reaction which leads to generation of nitrosated-amines and other related compounds cause alkylation. Also the bile acids released may be nitrosated to produce alkylating compounds. S-Adenosylmethionine, is one such kind of methylating agent known to induce mutations.

 

c) Exogenous alkylating agents: The major alkylating agents present in the environment are the atmospheric halocarbon, chloromethane. Apart from this the other halocarbons are iodomethane and bromomethane which are present in considerable quantities and are mutagenic and possibly carcinogenic. The other major contributors are tobacco nitroso compounds which are generated during the processing of tobacco.

 

d) Alkylating drugs: These comprise mainly of the anti-cancer drugs. These alkylating drugs are either methylating agents, e.g. temozolomide and streptozotocin, an antibiotic, or choloroethylating agents eg. carmustin, lomustine and fotemustine. These drug lead to lesions and interstand cross links.

The Ames test (to identify a potential carcinogen)

 

This test was developed by a biochemist, named Bruce Ames in the 1970’s to test the ability of a particular compound or chemical for its mutagenic potential. This particular test employs bacteria since they have short generation time to show the desired mutagenic effect. Hence a mutant strain of Salmonella was employed which lacked the ability to synthesize the amino acid histidine. Histidine is an important requirement for these bacteria, hence this mutant strain of bacteria will not be able to grow in a medium lacking histidine. This test aims to test the mutagenicity of a particular unknown compound by observing its potential to cause mutations reversion. In this case, the mutant revertants can now grow in a medium lacking histidine since they are able to synthesize their own. The more potent mutagen will lead to more number of colonies on the agar plate. Since liver is known to metabolize foreign substances and render them toxic (mutagenic), this test uses liver enzymes to identify compounds which can turn mutagenic in the liver.

b) Radiations

 

This is yet another cause of mutations. Radiations can be broadly divided into two categories (a) Ionizing radiations (X rays, gamma rays) (b) non ionizing radiations (UV rays).

 

Mutations arising from ionizing radiations : These radiations have both direct and indirect effects. The direct effects are the generation of single stranded or double stranded breaks. The breaks generated are not detected by repair system. During the indirect effect, the ionizing radiation interact with molecules in a cell to generate free radicals (ROS). Since the ROS have unpaired electrons, their chemical reactivity is quite high, thereby leading to mutations.

 

Mutations arising from non-ionizing radiations: The different ultraviolet (UV) wavelength components, UVA, UVB and UVC are known to possess different mutagenic potential.

 

a) UVA (320-400nm) – This type of radiations result in the formation of cyclobutane pyrimidine dimers, and also form oxidized DNA bases through an indirect mechanism. The dimerization of pyrimidine leads to lesions which cause DNA replication errors. The C → T transitions are most common followed by other transversions and frameshift type of mutations.

 

b) UV B (280-320nm) and UV C (200-280nm) – These type of radiations usually result in pyrimidine dimers and the pyrimidine photoproducts (6-4) as the major forms while purine dimers and pyrimidine mono-adducts as the minor forms. Photoproducts are formed by forming a stable bond at 6 and 4 positions between two neighboring pyrimidines and appear to form preferentially at 5′-TC and 5′-CC sequences.

Fig.6 (a) A cyclobutane pyrimidine dimer stimulates the formation of a four-membered cyclobutyl ring (green) between two adjacent pyrimidines on the same DNA strand (b) Structure of the 6-4 photo-product.

 

Mutations arising from base analogs and intercalating agents

 

Base analogs: These compounds mimic the natural bases and get incorporated in the DNA replacing normal bases to cause errors during replication. A classical example of a base analog is that of 5-Bromouracil (5-BU) which is an analog of thymine since it has bromo group at the C-5 position instead of a CH3 in the case of thymine. The mutation caused by 5-BU depends on its keto and ionized forms. Due to the presence or a bromo group the electron distribution keeps changing frequently which causes 5-BU to interchange from its enol form (which pairs with adenine) to its ionized form (which mistakenly pairs with guanine). Hence the ionized form of 5-BU lead to A.T → G.C transitions leading to mutations.

Another prominent base analog is 2-aminopurine which mimics adenine and base pairs with thymine but when its gets protonated it has a tendency to pair with cytosine, hence it causes A.T → G.C transition mutations.

  1. Base intercalation

This comprise of a class of compounds which adjust themse lves between the stacked nitrogen bases and modify them. These may lead to the addition or deletion of single or even a few nucleotide base pairs. These intercalating agents are basically flat molecules containing polycyclic rings. Examples of this class include proflavin, acridine and ethidium. Since the addition of even a single base pair may lead to changing of the reading frame of the entire length of the protein terminating till the carboxyl terminus, the mutations caused by such intercalating agents are called frameshift mutations. As a consequence, the entire sequence of amino acids coding for a particular protein changes, hence this results in loss of protein’s structure and thereby affecting its biological activity.

 

 

Consequences of mutations:

 

Kearns-Sayre syndrome – This syndrome occurs due to spontaneous mutations resulting in the deletion of a few nucleotide bases which lie in between the repeated sequences of DNA causing a dysfunctional mitochondrial phosphorylation resulting in altered mitochondrial structure.

 

Fragile X syndrome – This particular disease which causes mental retardation, actually results from trinucleotide repeat (CGG). Normal individuals have CGG repeats in the range of 6-54 copies but the affected individuals carry about 200-1300 of the repeat numbers. Hence the occurrence of this disease is related to 1 out of 1500 males and in 1 of 2500 females. Other diseases which are caused by a similar mechanism of expansion trinucleotide repeats are Muscular dystrophy, Kennedy disease , Huntington disease.

  1. Summary

This particular provided an insight into the world of DNA mutations , their causes, various categories under which the different types of mutations are grouped. We also tried to explain the repercussions of these mutational errors in brief.