October 02, 2013
Remember in Dune when Paul Muad’Dib took a sip of the “Water of Life” and needed weeks in a coma to turn it into something that let him survive and emerge even more powerful than before? Turns out yeast sometimes have to do something similar.
Now of course the yeast aren’t consciously moving molecules around to deal with a poison like Paul did. No, instead they sometimes need to transcribe low levels of a mutated gene over a long period of time to survive in a new environment.
This process is called retromutagenesis. The idea is that a cell gets a mutation that would allow it to survive and prosper in a new environment if only it could replicate its DNA. Unfortunately the new environment is so unforgiving that the cell can’t replicate.
The cell escapes this catch-22 by transcribing the gene with the mutation so that the mutant protein can get made. Once enough of this protein is made, the cell manages to get up enough steam to power through a cell cycle. Now the mutation is established and the yeast can make lots of mutant protein and happily chug along.
In a new study in the latest issue of GENETICS, Shockley and coworkers hypothesize that something like this is happening in their experiments. They were studying oxidative damage to DNA and found that some of their mutants required many days before they could grow in the absence of tryptophan (trp). They argue that these late arising revertants were due to the cells having to wait until retromutagenesis allowed enough functional Trp5p to be made so the cell could replicate.
The authors have created strains of yeast with various mutations in the TRP5 gene that cause the yeast to be unable to grow in the absence of trp. What makes these strains so useful is that they are set up in such a way that six different, specific point reversions can result in a functional TRP5 gene. They can then analyze any Trp+ revertants to see what types of damage lead to which type of mutations.
One of the first things the authors discovered was that oxidative damage caused all six different reversions. While this was interesting, the specific mutation they wanted to focus on was a G to T transversion which occurs when G is converted to 8-oxoguanine. This is why they focused on the trp5-A149C strain.
The main way that yeast cells deal with 8-oxoguanine is by removing it with the Ogg1 protein, a DNA glycosylase. When Shockley and coworkers deleted this gene in their strain, the number of revertants increased by 20-fold. From this they concluded that most of the revertants were the result of the misreplication of an 8-oxoguanine.
This is where the yeast run into a problem. In the absence of trp, the trp5 mutants do not replicate at all…they do not go through even one cell cycle. But to revert to a functional TRP5 gene, this strain needs to go through a cell cycle. This is why the authors think that the first step towards reversion is a mutation in the TRP5 transcript.
Consistent with this idea is the fact that the mutated G in this strain is on the transcribed strand and that this is important for high revertant frequencies. It also helps to explain why revertants took so long to appear. Basically there had to be a buildup of enough functional Trp5p to allow a single cell cycle to happen. Then the G could be converted to a T and the yeast could happily grow. In this specialized case, it looks like reversion is dependent on retromutagenesis.
But retromutagenesis, also called transcriptional mutagenesis, doesn’t happen only in yeast cells. It’s being studied as a possible way that all kinds of quiescent cells, such those in the process of becoming tumor cells, or bacteria whose growth has been stopped by an antibiotic, can mutate and escape the conditions that are restricting them. Our little friend may not save the human race from destruction like Paul did, but once again yeast is proving pretty darn useful in getting results that make a difference for human health.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics