Molecular mutations, means permanent alterations in sequences of nucleotides in the nucleic acid, which forms the genetic material. Mutation at the molecular level refers to any addition, deletion or substitution of the nucleotide bases in the DNA.
Mutations and Nucleotide Sequences in Nucleic acid
These alterations in base sequences may be of the following types:
1.Deletion of bases
2.Insertion of base
3.Inversion of a sequence and
4.Replacement of a base pair
Deletions, insertions and inversions include those changes in base sequences which involve breakage and reunion of DNA segments. However, replacement of a base pair may take place during replication of DNA without any breakage of DNA.
These base pair replacements can be of two types; transitions and transversions. Transitions are those base pair replacements, where a purine is replaced by another purine and a pyrimidine is replaced by another pyrimidine. Similarly transversions are those base replacements where a purine is replaced by a pyrimidine and vice versa.
These changes take place due to mistakes in the incorporation of nucleic acid precursors or due to mistakes during replication.
Effect of chemical mutagens on nucleotide sequence
Alterations during replication of nucleic acids
Incorporation of base analogues
5-bromouracil, 5-chlorouracil and 5-iodouracil can replace thymine in DNA. But 2-aminopurine incorporates in such a small amount that it is not possible to find out which base it replaces.
Although these base analogues are much less inhibitory for bacterial and phage growth than other purine and pyrimidine analogues, yet they are much more mutagenic.
5-bromouracil or 5-bromodeoxyuridine
It can pair with adenine just like thymine. Occasionally 5-bromouracil loses a hydrogen atom in position 1 and pairs with guanine instead of adenine. This pairing mistake may occur at the time of incorporation or after incorporation at the time of replication. Hence both kinds of transitions, G-C into A-T and A-T into G-C should be inducible.
The higher frequency of pairing mistakes are due to higher electronegativity of Br in Bu as compared to methyl group in thymine (methyl uracil). The pyrimidine ring gets poorer in electrons and thus can lose H- atom at position 1 more easily than in thymine, thus giving rise to enol form.
This suggests that mistakes in incorporation may be more frequent, since once incorporated, the electronegativity of Bu is reduced and the mistakes in replication will be less frequent. Thus changes GC→AT should be more frequent.
Bromodeoxyuridine is more effective mutagen probably because it easily converts to deoxyribosenucleoside triphosphate. Both bromouracil and bromodeoxyuridine induce point mutations.
2- Aminopurine (AP)
2- Aminopurine can pair with thymine by two hydrogen bonds and with cytosine by a single bond. The pairing involving two bonds is more common, since in the other case, nitrogen in AP and nitrogen in cytosine repel each other and cause their separation. Incorporation of AP at place of guanine to give a AP-C base pair will cause mutation in subsequent generations. Similarly a mistake in replication after incorporation of AP, leading to the formation of AP-T base pair may lead to a mutation. AP will thus induce transitions in both directions.
Inhibition of nucleic acid precursors:
There are several agents which interfere with normal synthesis of nucleic acid precursors. While some interfere with synthesis of purines or pyrimidines, others are more specific and interfere with the synthesis of thymine, adenine or guanine.
Azaserine is one of the strongest mutagen inhibiting purine synthesis. Since this is also an alkylating agent, its mutagenicity may be due to its alkylating ability rather than its inhibitory action.
Similarly urethane is an inhibitor of pyrimidine synthesis and also a mild alkylating agent. Thymine inhibits the production of chromosome breaks by urethane. It is possible that lack of one base either causes breaks or causes pairing mistakes. It is also possible that the inhibitor causes the formation of another base analogue which is the actual mutagen.
Alteration in resting nucleic acid
Nitrous acid deaminates, the bases G,C and A with decreasing frequency in DNA and with equal frequencies in RNA. Its mutagenic effect was analysed for tobacco mosaic virus (TMV), bacteria, phages, yeast and other organisms. HNO2 deaminates adenine to hypoxanthine, cytosine to uracil and guanine to xanthine. H pairs with C; U pairs with A and X with C. Thus HNO2 may bring about changes like AT to GC (due to deamination of A to H);GC to AT (due to deamination of C to U), but no change is brought about in the deamination of guanine to xanthine.
Hydroxylamine (HA) and hydrazine
In DNA, cytosine is the strongest reacting base when treated with hydroxylamine. Therefore it is possible that the major mutagenic effect of HA is due to alteration of C. Hydroxylamine probably causes hydroxylation of cytosine at amino group giving rise to hydroxylcytosine, which then subsequently pairs with adenine. This is because the hydroxyl-amino group should be more electronegative than amino group (due to electronegative oxygen) and hence this hydroxylated molecule should be more frequently in the tautomeric form having a hydrogen atom in the place of nitrogen at position-3. This tautomeric form cannot pair with guanine but can pair with adenine. Thus we should expect that the effect of hydroxylamine on DNA induces base pair transitions predominantly.
The effect of hydrazine lies in breaking the rings of uracil and cytosine giving rise to pyrazolone and 3-aminopyrasole respectively. Treatment of RNA by anhydrous hydrazine producers ribo-apyramidinic acid free of pyrimidines and the treatment of DNA produces the corresponding apyrimidinic acid.
Many mutagenic agents carry one, two or more alkyl groups in a reactive form. These are termed as mono, bi or polyfunctional alkylating agents. Most extensively studied alkylating agents both with respect to their chemical effects as well as mutagenic effects are diethyl sulphate (DES), dimethyl sulphate (DMS), methyl methane sulphonate (MMS), ethylethane sulphonate (EES) and ethyl methane sulphonate (EMS). They all act as monofunctional agents even when they carry two groups such as DES and DMS, since each group alkylates separately. Alkylating agents can alter DNA in four different ways.
1.Alkylation of phosphate groups of nucleic acids
When phosphate group is alkylated, unstable phosphate triester is formed, which hydrolyses to return the alkyl group. If enough alkyl groups remain attached till the time of duplication, the duplication might be inhibited. The attached alkyl group may, but not necessarily, interfere with replication in such a manner that non-complementary bases may be incorporated.
2.Hydrolysis of triester between sugar and phosphate
Hydrolysis of phosphate triester may take place between sugar and phosphate and thus causes the breakage of backbone. This will be lethal or cause larger alterations, but will not cause point mutations.
3.Alkylation of bases
Although 1 methyl adenine, 3 methyl adenine, 1-3 dimethyl adenine, and some cytosine derivative can also be obtained. 7 alkyl guanine is the most common derivative formed, which may pair with thymine instead of pairing with cytosine. The alkylated bases may inhibit DNA duplication and cause base pair mistakes during DNA duplication.
Alkylation of guanine at 7th position gives rise to quaternary nitrogen which is unstable. This either hydrolyzes away the alkyl group or else alkylated purine separates from deoxyribose sugar living it depurinated. The gap may then interfere with DNA duplication or may cause incorporation of wrong bases. The depurinated DNA is also more labile and may undergo breakage in the backbone soon after. This may induce large alterations or may be lethal but will not cause point mutations.