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This lecture covers about mutagens
- Chemical mutagens
- UV rays
- Transposons
Mutagens
Mutagens
Those agents which increase the mutation frequency in an organism is referred as mutagen. Mutagen may be physical, chemical or biological agents. This chapter, the common mutagens and their mode of action will be discussed.
A. Chemical mutagens:
A. Chemical mutagens:
Different classes of chemicals are involving in mutagenesis. There are three groups viz., base analogs, chemical modifiers and intercalating agents are found in the chemical mutagens.
1.Base analogs refers those molecules that resemble the purine and pyrimidine bases of DNA in structure yet display faulty pairing properties. If a base analog is incorporated into DNA in place of the natural base, the DNA may replicate normally most of the time. However, DNA replication errors occur at higher frequencies at these sites due to incorrect base pairing. The result is the incorporation of a wrong base into the new strand of DNA and thus introduction of a mutation. During subsequent segregation of this strand in cell division, the mutation is revealed. Examples: 5’ Bromouracil; 2-Aminopurine; Benzyl Amino Purine.
2. Chemical modifiers refers those chemicals induce chemical modifications in one base or another, resulting in faulty base pairing or related changes. For example, alkylating agents (chemicals that react with amino, carboxyl, and hydroxyl groups by substituting them with alkyl groups); deaminating chemicals (removal of amine group in nucleotides); depurinating chemicals (removal of purines from the DNA strand) are powerful mutagens and generally induce mutations at higher frequency than base analogs.
3. Intercalating agents are the planar molecules become inserted between two DNA base pairs and push them apart. Ex. Acridine orange. During replication, this abnormal conformation can lead to single base insertions or deletions in acridine-containing DNA. Thus, acridines typically induce frameshift mutations. Ethidium bromide, which is often used to detect DNA in electrophoresis, is also an intercalating agent and therefore a mutagen.
An introductory video about mutagens (Youtube)
Mode of actions
Mode of actions
5-Bromouracil
5-Bromouracil
It is a brominated derivative of uracil that acts as an anti-metabolite or base analog, substituting for thymine in DNA, and can induce DNA mutation. 5-BrU exists in two tautomeric forms that have different base pairing properties. The keto form is complementary to adenine, so it can be incorporated into DNA by aligning opposite adenine residues during DNA replication. Alternatively, the enol form are complementary to guanine. This means that 5-BrU can be present in DNA either opposite adenine or guanine.
Enol and keto forms of 5 Bromouracil had different mode of action to replace the nucleotides
When bU is in the cytoplasm, during DNA replication, it (keto form) replaced the thymine and pairs with A. During DNA replication (in next round), the bU, rarely changed its tautomeric structure to enol form in one strand pairs with G. In the subsequence replication, C:G will be in one genome. The original sequence was A:T, which was replaced by G:C, hence it is a transition type of point mutation (Base pair substitution).
2’Aminopurine
2’Aminopurine
2-Aminopurine, an analog of guanine and adenine. It most commonly pairs with thymine as an adenine-analogue, but can also pair with cytosine as a guanine-analogue and causes transition type of base pair substitution.
Nitrous acid
Nitrous acid
Nitrous acid is a chemical modifying mutagen, which is an oxidative deaminating chemical. It removes amine group from the nitrogen bases and thus induce the faulty pairing.
There are two common deaminations are:
a. Deamination of adeinine results in the formation of hypoxanthine. Hypoxanthine, in a manner analogous to guanine, selectively base pairs with thymine instead of cytosine. This results in a post-replicative transition mutation, where the original G-C base pair transforms into an A-T base pair.
b. Deamination of cytosine results in the formation of Uracil. Uracil always pairs with Adenine and results in the base-pair substitution of G:C to A:T.
Transition type of base-pair substitution induced by nitrous acid due to deamination of cytosine (to uracil, U) and thiamine (to hypoxanthine).
Apart from these, the nitrous acid can also deaminate guanine (to xanthine, which pairs with thiamine instead of cytosine) and 5-methylcytosine to thiamine and causes transition type of mutation.
Hydroxylamine
Hydroxylamine
Hydroxylamine can be used to mutagenize purified DNA. It results in C to T and G to A transition mutations when double stranded DNA is mutagenized. Hydroxylamine adds hydroxyl group to the nitrogen bases and thereby changes the base paring rules. The cytosine reacted with hydroxylamine and resulted into hydroxyl cytosine (HC), which pairs with A. Hence, C:G will be replaced by T:A (Refer the flow diagram).
Ethyl Methane sulphonate
Ethyl Methane sulphonate
Ethyl methane sulphonate is an alkylating agent, which adds methyl or enthyl groups in the N bases and thereby leads to mis-matching. For example, alkylation of Guanine leads to 6-Ethylguanine, which pairs with thymine instead of Cytosine. Likewise, alkylation of thymine by EMS forms 4-Ethylthymine, which pairs with Guanine instead of Adenine. Hence, the alkylation property of EMS leads to two types of base pair substitutions viz., G:C to A:T and A:T to G:C.
This will allow transition type of mutation
Depurination is an alteration of DNA in which the purine base (adenine or guanine) is removed from the deoxyribose sugar by hydrolysis of the beta-N-glycosidic link between them. EMS can able to perform depurination also. In such case, the depurinated nucleotide is recognized by PolIII (during replication) or (PolI during repair mechanism), it adds A in the corresponding complementary strand. Hence in subsequent generations, G:C pair will be replaced by T:A, in which Guanine is replaced by Thymine. Hence, depurination by EMS leads to transversion type of point mutation.
Intercalating agents look like base pair being inserted between nucleotides and thus causes addition mutation.
Watch youtube video describing different chemical mutagens and their function
B. Physical Mutagens
B. Physical Mutagens
Ultra violet rays
Ultra violet rays
As Gamma and UV rays are the physical agents that cause damage to the DNA. In contrast to UV radiation, ionizing radiation like gamma rays penetrates readily through glass and other materials. Therefore, ionizing radiation is used frequently to induce mutations in animals and plants because its penetrating power makes it possible to reach the gamete-producing cells of these organisms. However, because ionizing radiation is more dangerous and is less readily available than UV radiation, it finds less use in microbial genetics. UV rays are non-ionizing radiations extensively used to induce mutation in microorganisms.
The purine and pyrimidine bases of nucleic acids absorb UV radiation strongly, and the absorption maximum for DNA and RNA is at 260 nm. Killing of cells by UV radiation is due primarily to its effect on DNA. Although several effects are known, one well-established effect is the production of pyrimidine dimers, in which two adjacent pyrimidine bases (cytosine or thymine) on the same strand of DNA become covalently bonded to one another. The dimerization of adjacent base pairs (especially thymine) is mainly due to cyclobutyl ring formation (see the figure).
Due to dimerization, distortion of double helix will be formed (as shown in the figure) because, the base pairing with complementary strand will not be there is the dimer position.
Additionally the polymerization will also be blocked due to stalling of PolIII enzyme at this position. Additionally, DNA polymerase can also misread the sequence at this point.
The UV radiation source most commonly used for mutagenesis is the germicidal lamp, which emits UV radiation in the 260-nm region. A dose of UV radiation is used that kills about 50–90% of the cell population, and mutants are then selected or screened for among the survivors. If much higher doses of radiation are used, the number of surviving cells is too low. If lower doses are used, damage to DNA is insufficient to generate enough mutations. When used at the correct dose, UV radiation is a very convenient tool for isolating mutants and avoids the need to handle toxic chemicals.
Nice description about the mutagens (Youtube)
C. Biological mutagens
C. Biological mutagens
Transposable elements mediated mutagenesis is referred as biological mutagenesis. The same transposable elements present in phages (Mu phage) also have mutagenic effect. Unlike chemical or physical agent, the transposable elements will not do single base pair changes. A segment of DNA (about 1000-3500 bp) will be added in the genome. If the position of the genome has a gene, it will be inactivated due to the inserted sequence. Similarly, the chemical / physical mutagens are doing random mutagenesis in bacterial genome, while these transposable elements are site-specific, i.e., they will recognize the site at which insertion takes place. In this chapter, the structure and functions of transposable elements will be discussed.
Transposable elements are the DNA sequences that can change their relative position by themselves (self-transmissible) within the genome of a single cell. These sequences can be either “Copy and Paste” or “Cut and Paste” from one position to another position. The gene responsible for this codes an enzyme transposase, which mediates this process. This process is known as transposition. The transposition can create phenotypically significant mutations and alter the genome size.
Transposable elements are
- Stretches of DNA that can move from one site to another.
- Always found inserted into another DNA molecule such as a plasmid, a chromosome, or a viral genome.
- Do not possess their own origin of replications.
- Replicated when the host DNA molecule into which they are inserted is replicated.
- Having transposition ranging from 1 in 1000 to 1 in million.
- Wide spread nature and found in genome of all the three domains of life and also in virus and plasmids.
Types of transposable elements
There are two major types of transposable elements are found in bacteria viz., Insertion sequences (IS) and transposons (Tn). The common features of IS and Tn are
- Both carry genes encoding transposase, the enzyme necessary for transposition.
- Both have short inverted terminal repeats at their ends that are also needed for transposition.
The difference between IS and Tn is
- IS is a simple transposable element does not have any other genes whereas Tn carries some host genes (mostly antibiotic resistant genes) apart from its genes.
Insertion sequences: Insertion sequences are the simplest type of transposable element. They are short DNA segments, about 1000 nucleotides long and typically contain inverted repeats of 10–50 bp. Each different IS has a specific number of base pairs in its terminal repeats. The only gene they possess is for the transposase. Several hundred distinct IS elements have been characterized.
Transposons are larger than IS elements, but have the same two essential components: inverted repeats at both ends and a gene that encodes transposase. The transposase recognizes the inverted repeats and moves the segment of DNA flanked by them from one site to another. Consequently, any DNA that lies between the two inverted repeats is moved and is, in effect, part of the transposon. Genes included inside transposons vary widely. Some of these genes, such as antibiotic resistance genes, confer important new properties on the organism harboring the transposon. Because antibiotic resistance is both important and easy to detect, most highly investigated transposons have antibiotic resistance genes as selectable markers. Examples include transposon Tn5, which carries kanamycin resistance and Tn10, with tetracycline resistance.
Transposition
Both the inverted repeats at the ends of transposable elements and transposase are essential for transposition. The transposase recognizes, cuts, and ligates the DNA during transposition. During insertion into target DNA, a short sequence in the target DNA at the site of integration is duplicated. The duplication arises because single-stranded DNA breaks (like sticky end cut of Restriction endonuclease) are made by the transposase. The transposable element is then attached to the single-stranded ends that have been generated. The enzyme will repair by filling the gaps in the single stranded position, thus duplicated repeat will be formed at both the ends of transposable elements after transposition.
Mechanism of transposition
There are two types of transpositions viz., conservative transposition and replicative transposition.
Conservative transposition refers the excision of transposon from one position / location and reinsertion at second location, hence maintaining one copy only per genome. Ex. Tn5.
Whereas in replicative transposition, the transposon remains at its original site and one copy of it inserted in the new site during transposition.
Rarely, retro-transposition, which acts as retroviruses, through RNA and cDNA can do transposition.
Conservative transposition
Conservative transposition
Replicative transposition
Replicative transposition
Retro-transposition
Retro-transposition
Transposon Mutagenesis
When a transposon inserts itself within a gene, a mutation occurs in that particular gene. Mutations due to transposition do occur naturally. However, transposons are a conveniently used to create bacterial mutants in the laboratory. Typically, transposons carrying antibiotic resistance genes are used. The transposon is introduced into the target cell on a phage or plasmid that cannot replicate in that particular host. Using transposons, it is possible to get auxotrophic mutants also. Tn5 and Tn10 are the widely used transposons to create mutations in E. coli.
As that of mutation, transposition can also cause different effects.
Youtube video about transposition
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