Effect of radiations on nucleotide sequence

Ionizing radiations (X-rays, gamma (γ) rays, alpha (α) ray particles, protons, neutrons). The radiations can have direct effect on chromosomes. They may directly break chromosomes or alter one of the DNA bases or indirectly may initiate a chain of chemical reactions. The biological effect also depends on the kind of cell and stage of nuclear cycle. For instance, chromsomes are extremely sensitive to breakage in meiotic prophase. The frequency of mutants per viable organism often increases linearly with the dose. Timofeeff-Ressovsky et al. (1935) interpreted this relationship in terms of a target theory which states that single hit of the particle on the target (i.e. the genetic material) inactivates or mutates it. It is also assumed that each event occurs at random in an irradiated system, so that any target may be hit. Therefore, for small doses, the number of targets hit and the number of targets affected both will have linear relationship with amount of radiation. When the doses increase further, some of the hits may go waste, since they may hit a target already affected. Therefore, at higher doses the affected targets will be less than expected on the basis of linear relationship (Fig. 23.28). It is possible that radiations act through production of a chemical. The meaning of the word 'target' then depends on the nature of the chemical produced or metabolic lifetime of such a chemical i.e. whether it can get to any other place in the cell or is limited to the closest surroundings. 'Single hit' may then mean single chemical event or a single ionization or even a single cut by a densely ionizing particle.

The frequency of simple chromosomal aberrations e.g., terminal deletions, is proportional to the dose, since they arise by single hits. More complicated aberrations needing two breaks (interstitial deletions and exchanges), 'increase approximately with the increase of square of the dose (Fig. 23.29). The frequency of two hit aberrations is more if radiation is given continuously than rf given in fractions, since in the latter case healing of the breaks may take place. Intensity of radiation may also influence the frequency of aberrations, which would be higher at higher intensity and will increase with increase of square of the dose (Fig. 23.30). Chloramphenicol has been shown to keep the breaks open longer, showing a role of protein in healing.

The indirect effect of chemicals was obvious when certain chemicals were found to protect the organism from radiation and some other chemicals could reinforce the effect. For instance low O₂ concentration reduced the frequency of chromosome breaks induced by radiations. This oxygen effect (also known as anoxia effect) has been extensively studied and it is probable that radiations in the presence of O₂ form some peroxide radicals which may then influence the frequency of breaks and mutations. The peroxides may cause breaks or may prevent rejoining. Free radicals and peroxides are also believed to be formed along the path of radiations in cells and cause chromosome breaks or mutations. Ionization of water in cells may give free radicals and hydrogen peroxide in the following manner.



Non-ionizing radiations (ultraviolet rays). UV has more specific chemical effect than ionizing radiations, since UV absorption is limited to molecules carrying conjugated double bonds and each molecule has a special absorption spectrum with maxima at certain specific wavelengths. For nucleic acids, these wavelengths are in. the range 260-280 nm. UV of this range of wavelength exerts strong direct effect on nucleic acids.

In bacteria the possibility of an indirect induction of mutations by UV has been clearly established. UV irradiation of culture media have been found to produce mutagens which induced mutations in unirradiated organisms placed in these media within four hours. The radiation product is apparently unstable and organic peroxides or radicals have been suggested as the cause of mutation. Organic peroxides are mutagenic while H₂O₂ in saline is not. But since UV also acts on the pyrimidine bases and on the corresponding nucleotides within the bacterium, it is not known, how the mutagenic effect comes about. That most UV induced mutations involve extrachromosomal material is obvious from the fact that their frequency is reduced if post irradiation protein synthesis is blocked or altered. The frequency is similarly increased by addition of nucleic acid bases. The relationship of mutation frequency and UV dose is also not always linear or exponential.

For instance, in Aspergillus, at higher doses the frequency falls down (Fig. 23.31).

A relationship between UV and DNA could be shown by in vitro studies, where thymine and cytosine get hydrated by addition of a H₂O molecule (Fig. 23.32). Similarly two molecules of thymine get connected to give rise to a thymine dimer (Fig. 23.33).

Photoreactivation. An unusual fact about UV induced mutations was discovered by Kelner and others, who observed that UV effect can be reversed by exposing the cells to visible light containing wave lengths in the blue region of the spectrum. This phenomenon, known as photoreactivation, was observed in bacteria and bacteriophages and indicates that the damage caused by UV may be reversed before genetic material is permanently affected. Photoreactivation has been demonstrated to be caused by an enzyme whose specific activity seems to lie in its ability to split thymine dimers and repair DNA molecule. Other enzymes completely remove these dimers and repoiymerise missing nucleotides in E. coll. In this connection the. function of DNA polymerase I enzyme in E. coli. has been studied in detail for its function in repair of thymine dimers. In human beings also, the sunlight (due to UV light component) causes DNA damage, producing thymine dimers. However, due to the availability of a DNA repair system, UV damage is regularly and expeditiously repaired causing no problem. However, when this DNA repair system is impaired, as in patients suffering from skin prone disease, Xeroderma pigmentosum, the patient becomes susceptible to sunlight, because now it does not have the functional enzyme required for removing thyminc dimers produced due to UV damage. (For DNA repair, consult Chemistry of the Gene 2.  Synthesis, Modification and Repair of DNA).

Spontaneous mutations and nucleotide sequence
Spontaneous mutations are induced under normal conditions of growth. Some of these mutations should originate from mistakes in normal duplication of DNA. Transitions may be produced by tautomeric shift (or ionization) of bases, which leads to mistaken A-C base pairing (Fig. 23.34) and more frequently mistaken G-T base pairing (Fig. 23.35). The transitional changes in both directions can be obtained (Fig. 23.36). Transversions might arise by mistaken pairing between two purines or two pyrimidines. Several such pairs (A-G and C-T) are possible, using single or double bonds. In the latter case they have slightly wrong distances or angles for the two sugar (C)-base (N) bonds. Since DNA backbone is not completely stiff, it can accommodate such distortions, at least occasionally, as a mistake. AT→CG base pair changes may occur due to A-G or T-C base pairing (Fig. 23.37). Similarly GC→TA base pair changes will result from G-A and C-T base pairing (Fig. 23.38).


Deletions and additions may also occur spontaneously due to loop formation, when a base may not be copied or may be copied twice.