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Mutations - Raw Material for Bacterial Evolution

By Sophie Bolick
July 2010

Ivan Matic, Ph.D.
Speaking to many familiar faces, Matic said of his research, "If we want to look at selective pressures, we have to go back to nature or bring nature back to the lab." (Photo courtesy of Steve McCaw)

Richard Gradman, left, Dmitry Gordenin, Ph.D., center, and Anders  Clausen, Ph.D., right
Waiting to ask a question, Dmitry Gordenin, Ph.D., staff scientist with the LMG Chromosome Stability Group, center, listened attentively. Also pictured are lecture host Richard Gradman, left, and Anders Clausen, Ph.D., postdoctoral fellow in the DNA Replication Fidelity Group. (Photo courtesy of Steve McCaw)

Ivan Matic, Ph.D., a leading molecular geneticist with the French National Institute of Health and Medical Research in Paris, visited NIEHS June 7, as a guest of the Laboratory of Molecular Genetics (LMG) fellows. Matic spoke on the "Modulation of Mutation Rates in Bacteria," exploring the mechanisms of genetic alterations that play a key role in human disease causation and drive evolution.

The lecture was hosted by Postdoctoral Fellow Richard Gradman, Ph.D., a member of the LMG Spontaneous Mutation and DNA Repair Group.

Role of mutations in biological evolution

"Genetic viability is the basis for most evolutionary processes and is primarily produced by mutations," began Matic. Newly arising mutations are essentially neutral or deleterious and occur at higher rates than lethal or adaptive mutations. However, what is deleterious in a given environment can be adaptive in another environment.

There is a significant physiological cost attributed to the maintenance of the low mutation rates, due to the large number of proteins involved. There are numerous proteins that contribute to DNA replication fidelity in Escherichia coli (E. coli). DNA mismatch repair (MMR), which controls DNA replication fidelity, is one of the main DNA repair mechanisms a cell has to recognize and eliminate mismatched or unpaired bases in replicated DNA.

For this to occur, MMR differentiates between the DNA template strand, which is methylated, and the newly synthesized DNA strand, which is not. This process corrects 99 percent of replication errors in E. coli. As Matic demonstrated in a series of experiments (see text box), several proteins are involved in this process: MutS, which binds mismatched bases; MutL, which recruits MutH to the complex; and MutH, which cleaves the DNA.

Selective pressures and evolution

Optimizing selection takes into account a statistically continuous distribution. Individuals who have extreme values of trait have lower fitness than individuals in the middle of distribution.

To study this, Matic took 70 E. coli natural isolates and placed them under identical experimental conditions and measured their capacity to generate mutations. He found that mutation rates are not low and are highly variable. Nearly all strong mutators were MMR deficient mutants. "There must be strong selective pressure under certain conditions that favors mutation," reasoned Matic, because, according to theory, if an organism produces 100-fold more advantageous mutations, it is also producing 100-fold more deleterious and lethal mutations, so some of these strains should be dead, but they are not.

Experimental evolution in mice

The studies with the bacterial isolates were extended to germ-free mice colonized with the same bacterial strains and then continuously exposed to a series of antibiotics. Samples were taken every day, explained Matic, "So you can freeze evolution and go back and ask what's happening." Over time, the mutation rates increased. At the end of the experiment, there were 100 percent mutators.

Mutator populations are generating mutations to these antibiotics at high rates. What is happening in mice? "Mutators need smaller population size in order to generate new adaptive mutations than non-mutators. Hence, when selective pressure is very strong, you need multiple mutations in a small period of time in the same genome. A non-mutator cannot do this because he has no time."

Most mutator strains are mismatch repair deficient mutants

"Why is this?" asked Matic. "We don't necessarily have the answer, but I have a few ideas," he added. "First, MMR is not a DNA repair system, at least in bacteria. The mismatch repair system is eliminating replication errors. There is a reason you don't find DNA repair mutants that are mutators in the laboratory or in nature. If you're not repairing DNA lesions, you're dead. It's very dangerous. That's why you have all these natural MMR mutants."

"The second thing is MMR is not only controlling replication fidelity but also the fidelity of homologous recombination efficiency. MMR deficient alleles can hitchhike with mutations and recombination events," he continued. "One recombination event is less deleterious than one newly arising mutation, simply because with recombination you're reshuffling mutations that have already undergone natural selection."

(Sophie Bolick, Ph.D., is a postdoctoral fellow with the Molecular and Genetic Epidemiology Group in the Laboratory of Molecular Carcinogenesis.)

An experimental approach for detecting mutations

Matic fused green fluorescent protein (GFP) to the proteins MutL and MutS, respectively, and used them to detect mismatches in bacterial cells as visualized by fluorescent foci. These studies showed that there is 2- to 10-fold more MutL in cells than MutS per mismatch in the absence of MutH. Matic thinks that communication between mismatches and strand-discrimination site, the MutH cutting site, is achieved by multimers - proteins made up of more than one peptide chain - of MutL. These constructs are currently used as tools for studying molecular mechanisms of mismatch repair in his lab.

The next question Matic asked was "What is happening with the foci?" He found that any given foci disappeared within 40 minutes, which is the replication time for E. coli. The disappearance of foci indicates that the mutation was fixed at that moment. Additional experiments showed that the number of MutL-GFP foci was linearly related to the frequency of mutations.

"What you have here is the possibility for visualization of replication errors in live organisms," said Matic. "What's even more amazing is that method is allowing you to score most emerging mutations independently of their phenotype."

However, as Matic discovered, there are limitations to this assay. Certain mismatched bases, like a C-C mismatch, cannot be detected. If the clusters of mutations are too close, they are not distinguishable.

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