The combined action of sensitizers (S) and laser radiation (LR) on eukaryotic and prokaryotic DNAs has been analyzed in a number of works. The sensitizers used were ethidium bromide (EB), acridine orange (AO), and 8-methoxypsoralen (8-MOP). It has been shown that the possible mechanisms of break formation are those of radical formation, two-quantum affine modification of DNA bases, and others.
The objective of the present study was to examine the possible mechanism of DNA breakage and of chromosome aberration induction resulting from exposure to a new sensitizer, 6-mercaptopurine (6-MP), and LR (Burilkov et al., MNL 71:44-45, 1997). We have recently identified a sensitizer which, in combination with LR, enhances the mutagenic activity of AO, EB and 8-MOP by several fold. This is 6-mercaptopurine.
We have compared cytogenetic effects of the known sensitizers, AO and EB, and of previously unused ones, such as 6-mercaptopurine (6-MP) and Cloroxine (CX), each used in combination with laser radiation (LR). The studies have shown that the highest rate of chromosome aberrations occurred when 6-MP was used as a sensitizer. This exceeded the rates of chromosome aberrations resulting from exposure to EB+LR, CX+LR, and AO+LR by factors of 1.5, 4, and 8 respectively.
It has been suggested that one possible reason for multiple chromosome aberrations resulting from exposure to 6-MP and LR may be the formation of numerous additional breaks in maize genomic DNA due to laser energy which is transferred from a sensitizer molecule to certain DNA regions. During mitosis, these breaks may be repaired or they may become chromosome aberrations. To test the proposed hypothesis, genomic DNA of maize was studied by gel electrophoresis. Electrophoretic patterns in non-denaturing conditions and break counts from densitograms have shown our hypothesis to be a plausible one.
As a next step, it was deemed necessary to study some possible causes of DNA break formation on exposure to 6-MP and LR. One possible mechanism contributing to the induction of breaks could be that of radical formation. Indeed, the EPR spectra have confirmed the presence of radicals in DNA following its exposure to 6-MP and LR and their absence under no irradiation and with irradiation but in the presence of 2-mercaptoethanol (2-ME) known as a radical quencher .
However, the mechanism of DNA break induction due to radical formation is, in this case, not the only one. This is supported by electrophoretic patterns of DNA preparations exposed to LR in the presence of various concentrations of 2-ME. It is apparent that starting with a concentration of 0.1 M 2-ME, the number of DNA breaks is not reduced but, at the same time, it is considerably higher than that in the control samples. It should be noted that no radicals were present in the EPR spectra at a concentration of 0.1 M 2-ME.
Further and more detailed studies are needed to be able to provide an answer to the question of what other mechanisms contribute to the formation of DNA breaks. Part of the answer may be various mechanisms of DNA-S complex formation. At present, mechanisms for the formation of complexes of DNA with AO, EB, and 8-MOP are known. Intercalation of molecules of the indicated sensitizers between DNA base pairs results in some physicochemical characteristics of DNA (melting temperature, absorbance maximum, and others) being altered, which is consistent with our findings. For the DNA-6-MP complex, the melting temperature differs insignificantly from that of the DNA alone, and there is no shift in the maximum value of the absorption spectrum. These findings suggest that 6-MP, while interacting with DNA, alters insignificantly its structure and conformation, since 6-MP is similar in its structure to the DNA=D4 to a great extent, with the LR wavelength, DNA breaks can be supposed to be formed due to a direct transfer of the LR energy to certain, adenine-rich DNA regions.
In our view, the in vivo transfer of energy to the sugar-phosphate skeleton of DNA to form breaks and subsequent transformation of these into mutations can proceed according to two schemes:
- 6-MP, being homologous to adenine, forms complementary binary and ternary complexes with it in the sites of complete unwinding. LR drives the DNA-6-MP complex into an excited state which, in turn, results in DNA breaks and induces chromosome aberrations;
- during semiconservative DNA synthesis, 6-MP, being an adenine analog, can replace it at some sites, resulting in a modified DNA structure. In the sites of 6-MP localization in DNA, LR, on being absorbed by the sensitizer, induces breaks which may be repaired or may become chromosome aberrations.
In an in vitro system (DNA preparations and 6-MP), the first pathway
seems to be more plausible.
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