Laboratory Activity to Promote Student Understanding of UV Mutagenesis and DNA Repair.

INTRODUCTION

To maintain the integrity of the genome, DNA molecules need to be very stable. However, changes in DNA molecules are common, due to the effects of UV light, ionizing radiation, some types of environmental chemicals and some of the molecules produced by a cell’s own metabolism. To maintain the accuracy of the genetic code, cells have a variety of repair mechanisms. For example, the thymine dimers induced by ultraviolet light can be repaired by photorepair; the enzyme photolyase uses energy from visible light to break the abnormal bond in the dimer, restoring the DNA to its original normal structure (1, 2) (Fig. 1).

FIGURE 1

UV light can induce the formation of a thymine dimer. During photorepair, the enzyme photolyase uses energy from visible light to break the abnormal bond in the dimer and restore the DNA to its original normal structure.

This activity includes a low-cost, technically feasible experiment in which students observe the effects of UV mutagenesis and DNA photorepair in the archaeon Haloferax volcanii. Because this halophile grows on a very salty medium, there is no danger of contamination with pathogens and sterile technique is not required. The overall design of the activity (Fig. 2) mimics research that demonstrated photorepair in Halobacterium salinarum (3).

FIGURE 2

Design of experiment that demonstrates photorepair. Populations of H. volcanii are exposed to similar doses of UVC, and half of the populations recover in visible light so photorepair can occur.

An optional extension links this activity to topics of immediate concern to students—how exposure to UVC light contributes to skin cancer risk and the protective effects of sunscreen. Students design and carry out an experiment to test whether SPF 15 sunscreen increases the lethal exposure time for H. volcanii by a factor of 15.

Throughout the activity, discussion questions engage students in actively thinking about the biological phenomena and experimental procedures and analysis. The active learning in all phases of this activity has been found to increase student retention and understanding (4). This activity is designed for students in college or university genetics, microbiology or introductory biology courses, or high school honors biology courses.

PROCEDURE

Safety

We chose the halophile H. volcanii for this experiment because this high-salt requiring archaeon is harmless. Furthermore, no pathogens can grow on the high-salt (21%) agar that H. volcanii grows on. Therefore, while microbiology students may benefit from using sterile techniques and following ASM Biosafety Guidelines for this BSL1 (Biosafety Level-1) organism, experiments with H. volcanii are safe even without these precautions.

UV light can have harmful effects, so the UV light is mounted in a cardboard box, which students cover before they turn on the UV light (Fig. 3). In addition, students wear goggles and are instructed not to look at the lamp or expose their skin to UV radiation.

FIGURE 3

Protection from UVC light. The UV exposure box, made from a photocopy paper box and a clamp lamp, minimizes any risk of UV exposure for the students.

Materials

A detailed list of supplies and equipment is available in the Teacher Preparation Notes for “UV, Mutations, and DNA Repair” (see Appendix 1). The H. volcanii culture is available from Nasco (https://www.enasco.com/product/Z50319M). A kit including all materials needed to perform the experiment is also being developed by Nasco.

Experiments

Each student group spreads H. volcanii on their experimental plate and then exposes a quadrant of the plate to one of the specified durations of UVC exposure. Then, half the student groups wrap their plate in aluminum foil and all the plates are left near a window for one to three hours. The H. volcanii then grow for about three to five days in a 45°C incubator that teachers can make easily and cheaply or for two to three weeks at room temperature in the dark.

To determine whether photorepair has occurred, students estimate the lethal exposure time for plates with or without exposure to visible light after the UVC exposure. To estimate the lethal exposure time, students rate whether a lawn is present on quadrants with different durations of UVC exposure (Fig. 4).

FIGURE 4

Photorepair of UV-induced mutations increases survival of H. volcanii. Notice the increased survival of H. volcanii when 20, 30, or 40 seconds of UVC exposure was followed by one hour of sunlight exposure to allow for photorepair.

Additional information about the experimental procedure and the analysis and discussion questions are provided in the Student Handout (Appendix 2). A video that describes and demonstrates the experimental procedure is available at https://youtu.be/-g0OpR3NQKU. Additional information about required supplies and equipment, instructional suggestions, and background biology for teachers are provided in the Teacher Preparation Notes (Appendix 1).

The Student Handout and Teacher Preparation Notes also provide suggestions to help students design and carry out an experiment to evaluate the protection provided by SPF 15 sunscreen, together with information and questions that engage students in active learning about the relationship between mutations and cancer. These sections will be particularly appropriate for a genetics course and should probably be omitted for a microbiology course.

Note for high school biology teachers

Your students may benefit from the high school version of the Student Handout for this activity (Appendix 3) and a related activity that introduces students to the structure, function and replication of DNA (https://sites.google.com/site/molecularbiologyiw/home/haloferax-molecular-biology). The introductory activity includes an easy method for extracting DNA from H. volcanii. Teachers reported that “the introductory DNA activity gave a clear, concise and coherent explanation of DNA and DNA replication for the students” and “This activity allowed my students to see firsthand that all living things have DNA. The archaea are easy to maintain and safe for use in the classroom.” Both activities are aligned with the Next Generation Science Standards (http://www.nextgenscience.org/).

CONCLUSION

This activity provides an effective way to engage students in active learning about UV mutagenesis and DNA repair and reinforces student understanding of molecular biology and data analysis and interpretation. Thus far, this activity has been used successfully in several high school biology courses. Teachers report that the activity was valuable in helping students understand mutagenesis and photorepair and in developing student skills in designing and analyzing experiments.

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Click here for additional data file.