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Evelyn Witkin, Jean Weigle, the SOS Response and How E. Coli Generates Mutations in Response to UV Irradiation

Whether we will discover more examples of adaptive action by fascinating mutagenic enzymes is a question definitely on the 21st Century research agenda. Finding them would be an important contribution to contemporary evolution science.
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Last week, PLOS Genetics published a delightful interview with National Medal of Science winner Evelyn Witkin. The interview rewards the time it takes to read.

From the 1940s to the 1980s, Evelyn (born 1921) was a key pioneer in understanding how E. coli and other bacteria respond to UV radiation. Her fearless experiments into a totally unknown subject are a model for all young scientists.

Evelyn describes serendipitously discovering in her PhD research that UV killing was related to the control of cell division in the E. coli B strain she studied. At the time, no one imagined the connection she discovered. Today, we know that inhibiting cell division is a "checkpoint" control that prevents formation of daughter cells until genome damage has been repaired.

Subsequently, Evelyn worked on the complexities of cell responses to radiation damage. UV has two basic effects on E. coli: (1) it kills cells and (2) it causes an increase in mutations. Evelyn studied both effects and found that E. coli cells could repair both lethal damage and mutagenic damage. Her experiments indicated that the repair and mutation reversals were inducible functions activated by exposure to UV radiation.

Interestingly, there was evidence that the ability of UV to generate mutations was also an active inducible function of irradiated bacteria. The clearest experiments were not Evelyn's. They came from a Swiss physicist turned molecular biologist, Jean Weigle (1901-1968).

In the early 1950s, Weigle used the bacterial virus 位 (lambda) as a test system to explore UV lethality and mutagenesis. By studying killing and mutation of 位 virus, Weigle could separate the DNA being examined (inside the virus particles before infection) and the cell environment where killing, mutagenesis and repair all took place after infection.

Weigle systematically looked at all possible combinations of untreated and UV-irradiated 位 virus and host E. coli cells. He obtained the following results:

1. Untreated virus infecting untreated cells ==> no killing, basal virus mutations
2. Irradiated virus infecting untreated cells ==> killing, elevated virus mutations
3. Irradiated virus infecting irradiated cells ==> less killing, even more virus mutations
4. Untreated virus infecting irradiated cells ==> no killing, elevated virus mutations

Experiment 1 was the control situation, and experiment 2 confirmed that UV has both lethal and mutagenic effects on the 位 virus. These results were expected. The surprises (discoveries) came in the last two experiments.

Experiment 3 demonstrated that radiating the cells induced two different activities. One activity removed (repaired) lethal damage to the irradiated virus DNA. This activity was called "Weigle reactivation." The second activity increased the yield of mutations from irradiated DNA. This activity was called "Weigle mutagenesis."

Experiment 4 was the biggest surprise. Irradiated cells produced additional mutations on untreated viral DNA. This "untargeted mutagenesis" represented a totally unexpected inducible cell capacity to alter DNA sequences undamaged by radiation.

Evelyn recognized that UV DNA damage activates a coordinated cellular response. What we now call the "SOS response" comprises DNA repair and mutator functions as well as other changes in E. coli physiology, including the cell division inhibitor that Evelyn discovered in her PhD research.

Uncovering the exact molecular nature of the mutagenic activity of the SOS response involved my "adaptive mutation" colleague, Genevieve Maenhaut-Michel from Brussels. In 1986, she and a colleague spotted the role of the protein DinB in UV-inducible mutagenesis. "Din" stands for "damage-inducible", a name indicating DinB expression to be part of the SOS response.

Thirteen years later, DinB was recognized to be a new kind of DNA polymerase and renamed Pol IV. Polymerases I-III were already known from biochemical studies. Quickly, two other SOS proteins required for UV mutagenesis were also found to comprise a similar DNA polymerase named Pol V.

It became clear that Pol IV and Pol V were the Weigle mutagenesis functions in the SOS response. They represented a new class of mutagenic or error-prone polymerases that also had the ability to synthesize new DNA strands complementary to chemically damaged parental strands.

These error-prone "lesion-bypass" polymerases thus play a dual role in the radiation response. They allow replication to proceed on damaged DNA, and at the same time they introduce sequence changes (mutations) into the DNA copies.

The lesion-bypass polymerase sequence changes are not random. An important but little-noticed paper showed that each particular type of mutation in the newly synthesized DNA requires a specific trans-lesion polymerase or, in some cases, a pair of polymerases. If a required error-prone polymerase is removed from the cell, a particular kind of mutation does not occur in response to DNA damage.

What these results mean is that mutations are not just accidents that occur because damaged DNA is a poor template for new DNA chains. Special enzymes generate mutations on damaged DNA, and different trans-lesion polymerases generate particular kinds of mutations. The E. coli cell is in control of the mutagenic process through the particular polymerases it synthesizes.

Pol IV is responsible for certain cases of starvation-induced adaptive mutation in E. coli. A previous blog discussed the capacity of immune system cells to target and generate mutations for a specific function. Error-prone polymerases appear to play a role in that purposeful mutagenic activity as well.

Whether we will discover more examples of adaptive action by these fascinating mutagenic enzymes is a question definitely on the 21st Century research agenda. Finding them would be an important contribution to contemporary evolution science.

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