Chemical and Physical Mutagens

1. Chemical Mutagens:

These are the following types 

1. Base Analogues:

• A base analogue is a chemical compound similar to one of the four bases of DNA. 
• It can be incorporated into a growing polynucleotide chain when normal process of replication occurs.’ 
• These compounds have base pairing properties different from the bases. 
• They replace the bases and cause stable mutation.
• A very common and widely used base analogue is 5-bromouracil (5-BU) which is an analogue of thymine. 
• The 5-BU functions like thymine and pairs with adenine (Fig. 9.6A).
• The 5-BU undergoes tautomeric shift from keto form to enol form caused by bromine atom. 
• The enol form can exist for a long time for 5-BU than for thymine (Fig. 9.6B). 
• If 5-BU replaces a thymine, it generates a guanine during replication which in turn specifies cytosine causing G: C pair (Fig. 9.6A).

• During the replication, keto form of 5-BU substitutes for T and the replication of an initial AT pair becomes an A: BU pair (Fig. 9.7A). 
• The rare enol form of 5-BU that pairs with G is the first mutagenic step of replication. In the next round of replication G pairs with C. 
• Thus, the transition is completed from AT→GC pair.
• The 5-BU can also induce the conversion of GC to AT. 
• The enol form infrequently acts as an analogue of cytosine rather than thymine. Due to error, GC pair is converted into a G: BU pair which in turn becomes an AT pair (Fig. 9.7B). 
• Due to such pairing properties 5-BU is used in chemotherapy of viruses and cancer. 
• Because of pairing with guanine it disturbs the normal replication process in microorganisms.

• The 5-bromodeoxyuridine (5-BDU) can replace thymidine in DNA molecule. 
• The 2-amino-purine (2-AP) and 2, 6-di-amino-purine (2, 6-DAP) are the purine analogues. 
• The 2-AP normally pairs with thymine but it is able to form a single hydrogen bond with cytosine resulting in transition of AT to GC. 
• The 2-AP and 2, 6-DAP are not as effective as 5-BU and 5-BDU.

2. Chemicals Changing the Specificity of Hydrogen Bonding:

• There are many chemicals that after incorporation into DNA change the specificity of hydrogen -bonding. 
• Those which are used as mutagens are nitrous oxide (HNO2), hydroxylamine (HA) and ethyl-methane-sulphonate (EMS).

(a) Nitrous Oxide (HNO2):

• Nitrous oxide converts the amino group of bases into keto group through oxidative deamination. The order of frequency of deamination (removal of amino group) is adenine > cytosine > guanine.

(b) Deamination of Adenine:

• Deamination of adenine results in formation of hypoxanthine, the pairing behaviour of which is like guanine. 
• Hence, it pairs with cytosine instead of thymine replacing AT pairing by GC pairing (Fig. 9.8A).

(c) Deamination of Cytosine:

• Deamination of cytosine results in formation of uracil by replacing – NH2 group with -OH group. 
• The affinity for hydrogen bonding of uracil is like thymine; therefore, C-G pair­ing is replaced by U-A pairing (Fig. 9.8B).

(d) Deamination of Guanine:

• Deamination of guanine results in formation of xanthine, the later is not mutagenic. Xanthine behaves like guanine because there is no change in pairing behaviour. Xanthine pairs with cytosine. 
• Therefore, G-C pairing is replaced by X-C pairing.

(e) Hydroxylamine (NH2OH):

• It hydroxylates the C4 nitrogen of cytosine and converts into a modified base via deamination which causes to base pairs like thyamine. 
• Therefore, GC pairs are changed into AT pairs.

3. Alkylating Agents:

• Addition of an alkyl group to the hydrogen bonding oxygen of guanine (N7 position) and adenine (at N3 position) residues of DNA is done by alkylating agents. 
• As a result of alkylation, possibility of ionization is increased with the introduction of pairing errors. 
• Hydrolysis of linkage of base-sugar occurs resulting in gap in one chain.
• This phenomenon of loss of alkylated base from the DNA molecule (by breakage of bond joining the nitrogen of purine and deoxyribose) is called depurination. 
• Depurination is not always mutagenic. The gap created by loss of a purine can effectively be repaired.

Following are some of the important widely used alkylating agents:

(a) Dimethyl sulphate (DMS)

(b) Ethyl methane sulphonate (EMS) -CH3CH2SO3CH3

(c) Ethyl ethane sulphonate (EES) -CH3CH2SO3CH2CH3

• EMS has the specifity to remove guanine and cytosine from the chain and results in gap formation. Any base (A,T,G,C) may be inserted in the gap. 
• During replication chain without gap will result in normal DNA. In the second round of replication gap is filled by suitable base.
• If the correct base is inserted, normal DNA sequence will be produced. Insertion of incorrect bases results in transversion or transition mutation. 
• Another example is methyl nitrosoguanidine that adds methyl group to guanine causing it to mispair with thyamine. After subsequent replication, GC is converted into AT transition.

4. Intercalating Agents:

• There are certain dyes such as acridine orange, proflavine and acriflavin which are three ringed molecules of similar dimensions as those of purine pyrimidine pairs (Fig. 9.9). 
• In aqueous solution these dyes can insert themselves in DNA (i.e. intercalate the DNA) between the bases in adjacent pairs by a process called intercalation.

• Therefore, the dyes are called intercalating agents. 
• The acridines are planer (flat) molecules which can be intercalated between the base pairs of DNA; distort the DNA and results deletion or insertion after replication of DNA molecule. 
• Due to deletion or insertion of intercalating agents, there occur frameshift mutations (Fig. 9.10).

2. Physical Mutagens:

Radiations as Mutagens:

• Radiation is the most important among the physical mutagens. Radiations damaging the DNA molecules fall in the wavelength range below 340 nm and photon energy above 1 electro-volt (eV). 
• The destructive radiation consists of ultraviolet (UV) rays, X-rays, ү-rays, alpha (α) rays, beta (β) rays, cosmic rays, neutrons, etc. (Fig. 9.11).

• Radiation induced damage can be categorized into the three broad types: lethal damage (killing the organisms), potentially lethal damage (can be lethal under certain ordinary conditions) and sub-lethal damage (cells do not die unless radiation reaches to a certain threshold value). 
• The effect of damage is at molecular level.
• In a live cell radiation damage to proteins, lipoproteins, DNA, carbohydrates, etc. is caused directly by ionization/excitation, or indirectly through highly reactive free radicals produced by radiolysis of cellular water. 
• DNA stores genetic information’s so a damage to it assumes great dimension. 
• It can perpetuate genetic effects and, therefore, the cellular repair system is largely devoted to its welfare.
• When the bacteria are exposed to radiation they gradually lose the ability to develop colonies. 
• This gradual loss of viability can be expressed graphically by plotting the surviving colonies against the gradually increasing exposure time. 
• This dose-response graph is called survival curve. 
• The survival curve of bacteria is given in Fig. 9.12. 
• The survival curve is analysed by a simple mathematical theory called hit theory.

Ultraviolet (UV) Radiation:

• UV radiation causes damage in the DNA duplex of the bacteria and phages. 
• The UV rays are absorbed and cause excitation of macromolecules. 
• The absorption maxima of nucleic acid = (280 nm) and protein (260 nm) are more or less similar. 
• The DNA molecule is the target molecule for UV rays but not the proteins. However, absorption spectrum of RNA is quite similar to that of DNA.
• The excited DNA leads to cross-linking, single strand breaks and base damage as minor lesion and generation of nucleotide dimer as a major one. 
• Purines are generally more radio – resistant than the pyrimidine of the latter, thymine is more reactive than cytosine.
• Hence, the ratio of thymine-thymine (TT), thymine-cytosine (TC), cytosine-cytosine (CC) dimer (Fig. 9.14) is 10:3:3, respectively. 
• A few dimers of TU and UU also appear. 
• The initial step in pyrimidine dimerization is known to be hydration of their 4: 5 bonds.

• Formation of thymine-thymine (TT) dimer causes distortion of DNA helix because the thymines are pulled towards one another. 
• The distortion results in weakening of hydrogen-bonding to adenines in the opposing strand. 
• This structural distortion inhibits the advance of replication fork.

The X-Rays:

• The X-rays cause breaking of phosphate ester linkages in the DNA. 
• This breakage occurs at one or more points. Consequently, a large number of bases are deleted or rearranged in the DNA molecule.
• The X-rays may break the DNA either in one or both strands. 
• If breaks occur in both strands, it becomes lethal. 
• The DNA segment between the two breaks is removed resulting in deletion. 
• Since both the X-rays and UV rays bring about damage in DNA molecule, they are used in sterilization of bacteria and viruses.