DNA Damage Action Spectroscopy and DNA Repair in Intact Organisms: Alfalfa Seedlings

Abstract

Although sunlight is essential for plant life, the ultraviolet components in the solar spectrum pose potential threats to DNA-dependent metabolism (e.g., transcription of mRNAs for essential proteins) and to genetic integrity. To evaluate the risks to plant DNA of UV present in solar spectra reaching the earth today and in anticipation of increases in UV-B, we have developed methods for quantitating DNA damages at low, biologically relevant levels (to 1 lesion/2 million bases) in nanogram quantities (total requirement 100 ng/determination) of non-radioactive plant DNA.

In our method, plant tissue is embedded in agarose, non-DNA components digested enzymatically, and the DNA treated with an enzyme or other agent which specifically and quantitatively induces a single strand nick at each lesion site, while a companion DNA sample is incubated without the agent. After gel electrophoresis with molecular length standards under denaturing conditions, the DNA is renatured, stained with ethidium bromide and a quantitative electronic image obtained using a charge-coupled device-based camera system. The number of average molecular lengths of the treated and untreated DNAs are calculated, and the frequency of lesions computed.

This approach has allowed us to determine the efficiency of monochromatic radiation in the UV-B and UV-A range in inducing pyrimidine dimers in the DNA of irradiated intact alfalfa seedlings. Although UV-B is indeed quite efficient in damaging DNA in the seedlings, UV-A can also induce dimers in the DNA. Since today’s solar spectrum contains far more UV-A than UV-B, the damage from UV-A (calculated from the product of the efficiency of a given wavelength in damaging DNA times the number of photons at that wavelength in the solar spectrum) is a significant portion of the total damage inflicted by sunlight on this plant.

What are the biological consequences of such DNA damage? Since only unrepaired or misrepaired lesions pose biological problems for living systems, it is essential to know the ability of plant systems to cope with damages to their DNA. We are measuring repair of pyrimidine dimers in UV-irradiated seedlings in the dark and in the presence of visible light. Alfalfa seedlings employ both excision and photorepair for removal of pyrimidine dimers; however, the relative importance of these repair paths seems to depend strikingly on the initial damage level to the DNA of the seedling.