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
experiment’s 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 lab-
oratory investigations designed to enhance high school students’ understanding of bacterial DNA repair systems.
Key words: Ultraviolet radiation; Prodigiosin; DNA repair; Radiation resistance; Bacteria.

Introduction in order to motivate students and develop their awareness of

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 acti-
vation of a vast array of DNA damage-repair systems, which
have been thoroughly studied (Gelbart et al, 2000). The gen-
eration of mutations by radiation and their correction by
DNA repair systems have been recognised as important sub-
jects 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 molec-
ular) 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
genetics, students should first be exposed to various phenom-
ena 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 sub-
ject, combined with an active hands-on experience, will con-
tribute 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

30 JBE Volume 41 Number 1, Winter 2006


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 simi-
larities 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 bac-
terial 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.
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 100µl 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 30ºC. The growth
of Serratia bacteria in two different temperatures is designed
to create one pigment-producing culture (25ºC), and one
pigment-deficient culture (37ºC).
Preparation of plates
The cultures incubated overnight are then serially diluted 1:10
in sterile nutrient broth. This dilution is achieved by adding
100µl of the culture to 900µl 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 trans-
ferred 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 100µl 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
UV damage and bacterial DNA repair | Zion et al
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 differ-
ent 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 37ºC, or
for a few days at room temperature. The plates should then
be refrigerated until the next laboratory session.
Results
Serratia marcescens growth
Observing the S. marcescens plates immediately reveals differ-
ences in appearance.The 25ºC plate (Figure 2a) shows a growth
of red colonies, while the 37ºC 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
Volume 41 Number 1, Winter 2006 JBE 31
Zion et al | UV damage and bacterial DNA repair
Figure 2. Bacteria growth under the different conditions. S. marcescens (2a,
2b); E. coli (2c, 2d) at 25ºC and 37ºC 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 bacter-
ial 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 25ºC (Figure 2c) and the 37ºC (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 bor-
ders between individual colonies can be distinguished. On
the fourth section (i.e. 30 seconds), there was a growth of one
single colony at 37ºC, and of three colonies at 25ºC. 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
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 cul-
tures 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 speci-
fied 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 cellu-
lar death. This critical point can be observed in both bacter-
ial 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 pho-
tolyase, 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 mis-
match 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 cell’s 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 cell’s 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

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
cell’s desperate attempt to survive despite extensive damage
which already occurred. (Gelbart et al, 2000).
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.
Acknowledgements
We wish to thank Yosef Mackler for his editorial assistance.
Michal Zion’s 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 learn-
ing 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
UV damage and bacterial DNA repair | Zion et al
Bartlett W T, O’Donovan 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, 261-
264
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 long-
wave 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 molecu-
lar explanations of genetic phenomena. Journal of Biological
Education, 34, 200-205.
Morimoto K (2002) Demonstrating the influence of UV rays on liv-
ing 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 O’Donovan G A (1971) Effect of gamma
radiation on Serratia marcescens. Comparison of the radiosensitiv-
ity of pigmented and nonpigmented cells. Radiation Research, 48,
40-52.
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 60ºC.
A Petri dish usually requires 15-20ml of agar solution.
UV lamp supplier
Vilber Lourmat: a list of distributors can be found on the company’s
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.
Volume 41 Number 1, Winter 2006 JBE 33