Concept 28 Some types of mutations are automatically repaired.
Stan Rupert explains UV damage and light-activated DNA repair systems and Richard Setlow's work on thymine dimer repair.
Hi, I’m Stan Rupert. In the 1950s I got tired of doing physics and became interested in molecular biology. I started working with Solomon Goodgal at Johns Hopkins. Goodgal had heard about Albert Kelner’s work that showed a bacterial cell culture could mysteriously recover from apparent death by ultraviolet radiation. Kelner found that the cells only recovered when they were exposed to visible light. Placing culture samples from the light and dark treatments on culture plates showed that only the cells exposed to light could recover and grow. At the time, people thought that light destroyed "cellular poisons" created by the UV irradiation. Goodgal and I thought that cells recovered because light helped repair DNA damaged by UV. We tested our idea by measuring the "transformation" of Hemophilus influenzae bacteria. Transformation occurs when a bacterium grabs extracellular DNA, brings it through the cell wall, and integrates it into its own genome. The cell on the right has just been transformed with a piece of DNA containing a gene for antibiotic resistance, signified by the yellow circle. The transformed cell will express this new gene and survive when exposed to the streptomycin antibiotic. Using this gene for antibiotic resistance, we can determine which cells have been transformed by growing the cells on culture plates containing the streptomycin antibiotic. Transformed cells will be resistant to streptomycin and form colonies on the plate; cells that weren't transformed will die. We used this system to show that UV damaged DNA. We isolated DNA from a streptomycin-resistant strain of Hemophilus influenzae bacteria, irradiated one sample with UV, and used the second as a control. After putting the DNA into cultures of Hemophilus cells, we cultured the cells on streptomycin plates to look for transformants. Cells from the control culture received a working streptomycin gene and formed many colonies on the plate. However, few cells grew from the UV culture, we thought this must be because UV had damaged the streptomycin-resistant gene. But we had to rule out two other possibilities. The lack of growth in the UV culture could have been caused by something in the solvent that, after being altered by UV, prevented transformation. But we found this wasn’t possible, because normal DNA put into UV-irradiated solvent did not show any loss in transforming activity. The other possibility was that the UV-irradiated DNA did not integrate into the Hemophilus genome like unexposed DNA. This would have given us the same results, but for a different reason than our hypothesis. Instead of dying due to DNA damage, cells would have died because they never received the streptomycin-resistance gene. But we showed that UV-irradiated DNA is just as likely to integrate into the Hemophilus genome as normal DNA. We were left with the conclusion that the UV damaged the DNA and the streptomycin-resistance gene, though we didn’t know how. We then devised an experiment to test our theory that light helped repair the damaged DNA. We mixed the UV-damaged DNA with an extract from E. coli cells, and exposed one tube to light and one to dark. We hypothesized that light activated an enzyme in the E. coli extract, and the activated enzyme repaired the DNA damage. After several minutes, we removed the Hemophilus DNA from the E. coli extract and added it to a culture of streptomycin-susceptible Hemophilus cells. If repaired, the DNA should transform these cells into streptomycin-resistant cells. We found transformed cells in the light cultures, showing that a light-activated enzyme in E. coli fixes UV-damaged DNA. We had found the first known DNA repair system! Unfortunately for us, the enzyme responsible for the repair was present in so few copies we never isolated it. It was eventually identified 27 years later. Additional research showed that this enzyme – photolyase – repairs a specific type of DNA damage called thymine dimers. The dimers are damaging because they stop DNA replication. Dimers form when two adjacent thymines bind to each other instead of their complementary bases. UV light causes the dimers to form. Photolyase attaches to the dimer and breaks the thymine-thymine bond with energy from light. Not all organisms use photolyase to fix dimers. Richard Setlow and Bill Carrier discovered another system, called excision repair, that removes the dimer by removing a segment of the strand. The size of the segment depends on the organism. After the segment is removed, DNA polymerase fills in the empty space. DNA replication can also introduce errors to DNA. These errors are usually caught by the proofreading ability of the DNA polymerases. If the wrong base is incorporated, DNA polymerase stops and removes the base before continuing replication. Several other repair systems exist to repair errors that slip past the proofreader, as well as other types of damage besides dimers. DNA repair is an integral part of an organism's well-being.
Albert Kelner discovered photoreactivation after a frustrating year of unexplainable results. Kelner knew he was on to something, but his boss just thought he was sloppy.
Each cell in your body loses 10,000 bases per day due to spontaneous breakdown at 37 degrees Celsius. What would happen if our body temperature was much lower?