Zion et al | UV damage and bacterial DNA repair

Practical UV radiation damage and bacterial DNA repair systems

Michal Zion, Daniel Guy, Ruth Yarom and Michaela Slesak Science Teaching Center, School of Education, Bar-Ilan University, Ramat Gan, Israel

This paper reports on a simple hands-on laboratory procedure for high school students in studying both radiation damage and DNA repair systems in bacteria. The sensitivity to ultra-violet (UV) radiation of both Escherichia coli and Serratia marcescens is tested by radiating them for varying time periods. Two growth temperatures are used in order to induce the production of the melanin-like pigment prodigiosin in Serratia marcescens. Several explanations are then suggested for the differences observed, including cellular DNA repair systems and the presence of the intracellular pigment prodigiosin. The experiments results prove that the different sensitivities to radiation of both bacterial strains are caused by cellular DNA repair systems, and not by other cellular molecules, such as the pigment prodigiosin. The paper also suggests further laboratory investigations designed to enhance high school students understanding of bacterial DNA repair systems. Key words: Ultraviolet radiation; Prodigiosin; DNA repair; Radiation resistance; Bacteria.

Introduction
We live in a world full of external factors that can harm our cellular DNA, causing mutations that disturb and disrupt the cellular lifecycle (Gelbart et al, 2000). Radiation, either x-ray or ultra-violet, is one of the most common mutagens we encounter daily. For example, we are exposed daily to UV radiation from the sun. Defence systems against radiation damage are a necessity for all living cells, in order to minimise the harmful effects of UV radiation. Living cells are indeed equipped with various ways to minimise damage by the activation of a vast array of DNA damage-repair systems, which have been thoroughly studied (Gelbart et al, 2000). The generation of mutations by radiation and their correction by DNA repair systems have been recognised as important subjects in high school biology curricula (American Association for the Advancement of Science [AAAS], 1994; Barker, 1983; Lock, 1997). Unfortunately, in spite of the prevalence of UV radiation in our lives, most students have minimal awareness of it and the effects on living organisms (Morimoto,2002). UV radiation and its effect on DNA were found to be particularly difficult subjects to learn, and students found this material too abstract (Johnstone and Mahmoud, 1980; Hallden, 1988; Pashley, 1994; Bahar et al, 1999). Furthermore, research has shown that students find the micro level (cellular and molecular) difficult to understand (Marbach-Ad and Stavy, 2000). One reason for the difficulties students encounter is the fact that the micro levels are generally taught in a theoretical manner, while the processes and components at these levels cannot be touched or directly observed (Marbach-Ad and Stavy, 2000). Marbach-Ad and Stavy (2000) suggested that 30 JBE Volume 41 Number 1, Winter 2006

in order to motivate students and develop their awareness of genetics, students should first be exposed to various phenomena in human beings or other primates and mammals, only in macro level terms. However, in examining micro levels, or in attempting to link the macro level with the micro level, it is only possible to work with lower organisms such as bacteria. This article suggests a simple laboratory protocol which provides middle and high school students (9-12th grades) with a hands-on activity demonstrating radiation damage. The protocol examines the activity of DNA repair systems by using two strains of bacteria that differ in their resistance to UV radiation. We believe that a relevant visualisation of the subject, combined with an active hands-on experience, will contribute to students deeper understanding of these subjects.

The effect of UV radiation on DNA

The effects of UV radiation, absorbed mainly from sunlight, have been thoroughly investigated for many years. These investigations found that UV radiation is absorbed mainly by the nucleotides in the DNA (Gelbart et al, 2000). The influence of this absorption is the bonding of two adjacent thymine bases on the same DNA strand, creating a structure called a thymine dimer. These dimers cause a distortion of the DNA molecule, and therefore cause malfunctions in the replication of the cell, potentially leading to cell death in unicellular organisms. In multicellular organisms, the damage caused by radiation may also lead to cell death, but can also cause severe diseases, such as the development of tumours and cancer (Gelbart et al, 2000). In order to protect themselves against UV radiation, organisms have developed a vast array of repair and protection mechanisms. For example, the pigment

UV damage and bacterial DNA repair | Zion et al

melanin in humans and mammals (and anthocyanins in some plants) acts as an external protection agent (Delpech, 2000). The molecules of these agents absorb the UV radiation and prevent it from reaching the genetic information in higher organisms cells. Other mechanisms repair the damage caused by radiation to the cellular DNA. Gelbart et al (2000) described the major DNA repair systems in bacteria: photo-reactivating enzyme (photolyase) excision repair through the Ultra Violet radiation ABC system (UvrABC), post replication repair, and SOS repair. These systems are described in the Discussion below. The laboratory protocol suggested here offers a method of comparing the resistance of two strains of bacteria to UV radiation, and of evaluating several factors that influence this resistance. The protocol makes use of two strains of bacteria: Escherichia Coli (E.Coli) and Serratia marcescens (S. marcescens). The most prominent difference between the two is that S. marcescens produces a red pigment called prodigiosin, whereas E.Coli colonies are white. Prodigiosin has structural similarities to a side chain in the human melanin molecule. These similarities have led researchers to hypothesise that prodigiosin may protect S. marcescens from UV radiation, just as melanin protects humans (Carter et al, 1976; Kaidbey and Kligman, 1978). The protocol examines the resistance of the two bacterial strains to UV radiation. In one test group, the production of prodigiosin is neutralised in order to investigate the source of the observed differences: either pigmentation or DNA repair mechanisms. a wave length of 245nm. In order to sterilise the radiation area and allow the lamp to reach a stable state, we suggest that the lamp operate for at least 30 minutes before irradiating the Petri dishes in the lab. The optimal distance between the UV lamp and the dish is 25-30cm. The bottom of each Petri dish should be divided into six equal sections using a felt-tip marker. Each of these sections will be irradiated for a different amount of time. The number of seconds is marked on the bottom border of each section: 0, 3, 10, 30, 60 and 120 seconds. A cardboard circle, the size of the Petri dish, minus a section the size of one sixth of the plate, is prepared. The Petri dish is then placed on a plastic/cardboard tray under the UV lamp, to ensure that the students hands will not be exposed to the radiated area. The dish should be open and covered with the cardboard so that only one section of the plate is exposed to the UV radiation, as shown in Figure 1. Take care that the cardboard is only slightly larger than the Petri dish, so that the cardboard does not touch the surface of the agar. Remember to use UV protection goggles, and to aim the UV lamp toward the radiation area only during the time allocated. Every section on the Petri dish should be exposed to UV rays for a varying amount of time: 0, 3, 10, 30, 60 and 120 seconds, as indicated on the bottom of the dish. Move the cardboard circle to another section only when the plate is outside the radiated zone. Ensure that the UV lamp remains on during the entire experiment. After irradiating each plate, the plates are incubated upside-down for 24hrs at 37C, or for a few days at room temperature. The plates should then be refrigerated until the next laboratory session.

Materials and methods

Preparation of bacterial cultures A commercially prepared culture of each bacterial strain (E.Coli and S. marcescens) is initially diluted and replenished, by adding 100l of the commercially prepared culture into 5ml nutrient broth (see Appendix for recipe) in 50ml test tubes. The dilution should be performed in test tubes containing an air volume at least nine times higher than the liquid volume, in order to allow a sufficient oxygen supply during the growth of the bacterial cultures. Two E.Coli cultures and two S. marcescens cultures should be prepared, and then incubated overnight in a water bath, at two different temperatures. The pigment prodigiosin does not develop in temperatures higher than 30C. The growth of Serratia bacteria in two different temperatures is designed to create one pigment-producing culture (25C), and one pigment-deficient culture (37C). Preparation of plates The cultures incubated overnight are then serially diluted 1:10 in sterile nutrient broth. This dilution is achieved by adding 100l of the culture to 900l of nutrient broth. Each culture should be diluted in this manner to produce a total of four diluted bacterial cultures. The diluted cultures are then transferred and spread on separate nutrient agar Petri dishes. A glass spreader, sterilised by dipping it into 95% ethyl alcohol and passing it in a flame, is used to spread 100l samples of culture on the agar surface. Alternatively, sterilised glass balls can be used to spread the sample on the agar. UV radiation UV irradiation is performed using a bactericide UV lamp (such as the Vilber Lourmat VL-6LC UV lamp - see supplier) with

Results
Serratia marcescens growth Observing the S. marcescens plates immediately reveals differences in appearance.The 25C plate (Figure 2a) shows a growth of red colonies, while the 37C plate (Figure 2b) is occupied by white colonies. In both plates, a massive bacterial growth appears in the first three sections (i.e. 0, 3, and 10 seconds
Figure 1. The UV irradiation procedure

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Zion et al | UV damage and bacterial DNA repair

for a maximum of 10 seconds, yet S. marcescens was still vital after 30 seconds, and even presented growth of individual colonies after 60 and 120 seconds. The growth of E.Coli cultures in two growth temperatures (likewise performed with S. marcescens) serves as a control experiment, showing that the differences found in radiation resistance between the two bacterial strains are not temperature dependent. It is therefore proposed that the difference in UV resistance may result from a greater efficiency of the DNA repair systems in the S. marcescens strain than in E.Coli. A closer examination of the results reveals that the growth delay does not occur in direct relation to the increase of radiation time. After a specified radiation time, a sharp decrease occurs in the survival rate (10 seconds in E.Coli and 30 seconds in S. marcescens). This phenomenon is explained by the work of DNA repair systems. As long as the repair systems are able to minimise radiation damage, the survival rate will be quite high. However at some critical point, the radiation damage reaches a level that the DNA repair systems cannot manage. This leads to an increase in the mutation rate and therefore to increased cellular death. This critical point can be observed in both bacterial strains (E.Coli between 10 and 30 seconds; S. marcescans between 30 and 60), and indicates involvement of the DNA repair systems. DNA repair systems Living cells have evolved a vast array of enzymatic systems in order to repair DNA damage. As described above, the primary damage caused by UV radiation is the formation of thymine dimers. An enzyme called photoreactivating enzyme, or photolyase, binds to the dimer and splits it into two normal pyrimidine bases. The action of the enzyme occurs only in the presence of certain wavelengths of visible light, and therefore does not occur in the dark. This enzyme has been found in bacteria (but not in humans) and is responsible for a significant amount of the repair of UV damages (Gelbart et al, 2000). A more complex repair system is termed excision repair. In this case, the products of the genes uvrA, uvrB and uvrC create the endonuclease enzymes UvrA, UvrB and UvrC, which together with the enzyme helicase are involved in cutting a strand of nucleotides from the mutated area. After removing the damaged strand, the gap is filled by repair synthesis, and is sealed by the enzyme DNA-ligase. During the DNA replication, and because of specific mutagens, some mismatching may occur. Mismatching refers to the matching of two wrong nucleotides in the DNA. This mismatch causes a structural distortion of the double helical DNA, and is recognised by unique enzymes. These enzymes remove the wrong nucleotide from the new DNA strand, and then insert the correct base. This repair mechanism is called mismatch repair (Gelbart et al, 2000). Sometimes DNA repair cannot be performed before or during the replication process. In such cases DNA replication ceases at the error site, skips it, and resumes just past the site. After replication, the recA gene plays a role in repairing the damage by sealing the gap with DNA, cut from the cells sister DNA molecule. This process is accompanied with an increase in the recombination rate. As a last resort, the bacterial cell uses the SOS repair system. This repair system is activated only in cases in which the cells life is at stake. When extensive DNA damage has occurred because of mutagens, and the damage is too extensive for other systems to repair, the replication

Figure 2. Bacteria growth under the different conditions. S. marcescens (2a, 2b); E. coli (2c, 2d) at 25C and 37C respectively.

respectively). The fourth section (30 seconds) in both plates demonstrates a significant bacterial growth, but the borders of individual colonies may be distinguished. The fifth and sixth sections (60 and 120 seconds) are almost sterile, with only the growth of isolated individual colonies. The lines of bacterial growth found on the borders of sections 4-5 and 5-6 on both plates exist due to overlapping of covered sections during UV radiation. They have no relevance to the results discussed. Escherichia Coli growth Both the 25C (Figure 2c) and the 37C (Figure 2d) E.Coli plates demonstrate a massive bacterial growth on the first two sections (i.e. 0 and 3 seconds respectively). The third section (10 seconds) shows significant bacterial growth, but the borders between individual colonies can be distinguished. On the fourth section (i.e. 30 seconds), there was a growth of one single colony at 37C, and of three colonies at 25C. The remaining sections are completely sterile. In addition, there is no difference in colour between the two E.Coli plates.

Discussion
Some researchers suggested that the red pigment in the S. marcescens cells (called prodigiosin) could contribute to its greater resistance against radiation (Bartlett et al, 1970; Hand and Mroz, 1971; Webb et al, 1971). This might be because of its structural similarities to melanin - a pigment that provides partial protection to animals and humans against UV rays. The comparison between the sensitivities of both pigmented and non-pigmented S. marcescens strains (accomplished by the use of two different growth temperatures) allows us to evaluate the role of prodigiosin as a protection agent against UV radiation. Figures 2a and 2b demonstrate that prodigiosin does not change the sensitivity of S. marcescens bacteria to UV rays, because there is no difference in resistance between the prodigiosin positive plates (Figure 2a) and the prodigiosin negative plates (Figure 2b). By comparing the two bacteria used in this experiment, we conclude that E.Coli bacteria are less resistant to UV radiation than S. marcescens; E.Coli bacteria survived radiation 32 JBE Volume 41 Number 1, Winter 2006

UV damage and bacterial DNA repair | Zion et al

process ceases. This is the stage in which the SOS system begins its action. The SOS system is capable of replicating opposite thymine dimers, and does so by inserting non-specific bases into the DNA strand. Because this process is not guided by a template-strand, the process causes a broad spectrum of mutations, which can prove fatal. The SOS repair system is the cells desperate attempt to survive despite extensive damage which already occurred. (Gelbart et al, 2000).
Bartlett W T, ODonovan G A and Neff R D (1970) Effect of gamma radiation on Serratia marcescen. Studies on the radiosensitivity of prodigiosin production. Radiation Research, 43, 196-203. Carter D M, Condit E S and London D A (1976) Effect of pigment on photomediated production of thymine dimmers in cultured melanoma cells. The Journal of Investigative Dermatology, 67, 261264 Delpech R (2000) The importance of red pigments to plant life: experiments with anhtocyanins. Journal of Biological Education, 34, 206-210 Gelbart W M, Lewontin R C, Suzuki D T, Miller J H and Griffiths A J F (2000) An Introduction to Genetic Analysis. 7th ed. New York, USA. W H Freeman and Company. Hanks A R and Mroz E (1971) Ultraviolet radiation sensitivity of white mutants and red wild-type Serratia marcescens. Radiation Research, 48, 312-318 Hallden O (1988) The evolution of the species: Pupil perspectives and school perspectives. International Journal of Science Education, 10, 541-552 Johnstone A H and Mahmoud N A (1980) Isolating topics of high perceived difficulty in school biology. Journal of Biological Education, 14, 163-166. Kaidbey K H and Kligman A M (1978) Sunburn protection by longwave ultraviolet radiation-induced pigmentation. Archives of Dermatology, 114, 46-48 Lock R (1997) Post-16 Biology Some Model Approaches? School Science Review, 79, 33-38 Marbach-Ad G and Stavy R (2000) Students cellular and molecular explanations of genetic phenomena. Journal of Biological Education, 34, 200-205. Morimoto K (2002) Demonstrating the influence of UV rays on living things. Journal of Biological Education, 37, 39-43. Pashley M (1994) A chromosome model. Journal of Biological Education, 28, 157-161. Webb P S, Neff R D and ODonovan G A (1971) Effect of gamma radiation on Serratia marcescens. Comparison of the radiosensitivity of pigmented and nonpigmented cells. Radiation Research, 48, 40-52.

Conclusions
This study introduces a simple experiment that examines the effects of UV rays on two strains of bacteria. The experiment can be easily performed in the school laboratory, and its results can be clearly observed after a short time. This experiment can also be the first in a series of experiments, testing for various factors that affect bacterial resistance to radiation. Students can develop their own ideas for further testing. Suggestions include: the influence of light intensity on the survival rate of bacteria (the photolyase enzyme is light-dependent and should prove more efficient when the bacterial cultures are placed in the light) the effect of light present in the radiation chamber the effect of incubating the Petri dishes in different light intensities the effects of different wavelengths on the survival rate of bacteria. In conclusion, we believe that the proposed experiment enables high school students to meaningfully understand the subject of UV radiation damage and DNA repair systems by implementing their theoretical knowledge via hands-on activities. This experimental system may serve as a basis for further open inquiry in the future.

Appendix
Solutions and Media Nutrient broth Peptone 10.0g, sodium chloride 5.0g, yeast extract 5.0g, boiled in distilled water to a final volume of 1000ml, then sterilised in an autoclave for 60 minutes. Nutrient agar Before sterilising, add 15.0-20.0g of agar-agar into the nutrient broth. Bring to a boil, and then sterilize the solution as directed. Pour the sterilised agar solution into the dishes as soon as it cools to 60C. A Petri dish usually requires 15-20ml of agar solution. UV lamp supplier Vilber Lourmat: a list of distributors can be found on the companys website: www. vilber.com Michal Zion (corresponding author) is a lecturer in the School of Education, Bar-Ilan University, Ramat-Gan, Israel 52900. Email: zionmi@mail.biu.ac.il Tel: + 972-50-8807365. Fax: + 972-8-9265476.

Acknowledgements
We wish to thank Yosef Mackler for his editorial assistance. Michal Zions work was supported by the Sacta Rashi Foundation. This research was supported by Uri and Ruth Oppenheimer`s contribution in memory of Ruth and Zvi Oppenheimer.

References
American Association for the Advancement of Science, Project 2061 (1994) Benchmarks for science literacy. New York, USA. Oxford University Press. Bahar M, Johnstone A H and Hansell M H (1999) Revisiting learning difficulties in biology. Journal of Biological Education, 33, 84-86 Barker G R (1983) Update: Biochemistry of Genetic Manipulation. Journal of Biological Education, 17, 101-04

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