The following points highlight the three main stages for mechanism of induced mutagenesis. The stages are: 1. Base Analogue Incorporation 2. Base Alteration 3. Distortion of DNA.
Stage # 1. Base Analogue Incorporation:
Some chemical compounds are sufficiently similar to the nitrogen bases of DNA so that they occasionally are incorporated into DNA in place of normal bases, such compounds are called ‘base analogues’. These base analogues, once placed in DNA, have pairing property unlike those of replaced bases and cause mutation by insertion of wrong nucleotides opposite to them during replication.
Commonly found pyrimidine analogue is 5-bromouracil (5 BU) or 5-bromodeoxyuridine (5 BUdR) which can pair with purines, i.e., adenine and guanine. Purine analogues include 2-amino- purine (2 AP) which can pair with pyrimidine such as cytosine and thymine (Figs. 13.6, 13.7).
Stage # 2.Base Alteration:
Some mutagens are not incorporated into DNA, but instead alter a base causing mispairing. Such alterations involve alkylation, depurination, deamination, hydroxylation, etc.
This is caused by alkylating agents include ethyl methane sulphonate (EMS), diethyl sulphonate (DES), nitrosoguanidine (NG), dinnethyl sulphonate (DMS), etc. Alkylation of bases involves addition of alkyl group, an ethyl group in case of EMS and a methyl group in case of DMS, to many positions of all four bases.
Alkylation of guanine leads to direct mispairing with thymine and would result in G—C→A—T transitions at the next round of replication (Fig. 13.8A).
Alkylation of bases or phosphates may cause hydrolysis of sugar-base linkage or sugar- phosphate linkage releasing the base from DNA molecule or breaking the backbone of DNA. This creates gap in chain and insertion of incorrect base results intrans-version or transition (Fig. 13.8B).
Alkylation of purine gives rise to unstable quaternary nitrogen leading to interruption of glycosidic bond between base and deoxyribose. The subsequent loss of purine residue from DNA makes it depurinated. These apurinic site cannot specify a base complementary to the original purine during DNA replication and may cause incorporation of wrong base.
This will lead to base pair substitution which could be either transition or trans-version. The depurinated DNA is also more labile and may undergo breakage in the backbone (Fig. 13.9).
This is caused by chemical like nitrous acid. Nitrous acid (HNO2) reacts with bases containing aminos group is replaced (deamination) to cause altered pairing (table 13.4). The order of frequency of deamination is – adenine > cytosine > guanine.
Mutagen like hydroxy- lamine(NH20H) is very specific in its action and reacts mainly with cytosine bringing about transition and mispairing. It causes hydroxylation of cytosine at amino group giving rise to hydroxyl cytosine (HC) which pairs with adenine instead of guanine. Thus C-G pairing is changed to A-T pairing (Fig. 13.11).
Stage # 3.Distortion of DNA:
The structure of DNA is distorted by radiation and intercalating agents.
(a) Intercalating agents:
This is another important class of DNA modifier. This group of compounds includes acridine orange, proflavin. These can mimic base pairs and are ablsto slip themselves intercalate between the stacked nitrogen bases at the core of DNA double helix (Fig. 13.12). This distorts the structure of DNA and can result in deletion or insertion of bases during replication.
High energy ionizing radiation and non-ionizing UV light are important mutagenic agents, causing distortion of DNA.
(i) UV rays (Dimerisation):
Both DNA and RNA preferentially absorb UV light causing their nitrogen containing bases to become highly reactive free radicals. The resulting un-stability causes the conversion of one base to another. The two major products of UV absorption appears to be pyrimidine dimers and pyrimidine hydrates.
Primary mutagenic effect of UV light appears due to production of thymine dimer (Figs. 13.13). The unsaturated bonds of adjacent pyrimidine’s become covalently linked to form a ‘cyclobutane’ ring. Irradiation of bacterial culture and subsequent extraction of DNA yields three possible types of pyrimidine dimers in DNA — thymine-thymine (50%), thymine-cytosine (40%) and cytosine-cytosine (10%).
Pyrimidine dimers can also be formed between adjacent strands, in RNA, pyrimidine dimers are formed between adjacent uracil and cytosine rings. Pyrimidine dimers cannot fit into the DNA double helix and cause distortion of the molecule. If the damage is not repaired, replication is blocked, leading to lethal effects.
UV radiation also causes addition of water molecules to pyrimidine’s in both DNA and RNA resulting in the formation of photo-hydrates (Fig. 13.13c).
X-rays bring about mutations by breaking the phosphate ester linkages in DNA. The breakage may take place at one or more points. As a result a large number of bases are lost or rearranged. In double stranded DNA, breaks may occur in one or both strands. Only the latter type is lethal. The biological effects of X-ray may be both direct and indirect.
Direct effect (Target theory):
The radiation energy is directly transferred to an atom or molecule of genetic material. The hit of the particle on the genetic material mutates it.
Indirect effects (Chemical theory):
The major genetic effects of radiations is that their energy is involved in the production of free radicals from water .Theses free radicals then reacts with DNA to after its structure .
Some chemicals produce same kinds of mutagenic effect that X-rays produce, hence are called radiomimetic chemicals.