March 19, 2014
Life is a set of tradeoffs for people, countries, and even cells. For example, governments need to decide how much money to dedicate to defense and how much to economic growth. Too much on defense and your country fails, because defense spending sucks up so many resources that your country can no longer afford to pay for anything else. And of course if you spend too little on defense, someone who spent a bit more can come and take you over.
No country lives in a vacuum though—how much to spend on defense and how much on growth depends on the country’s situation. If you are the Ewoks living next to an Imperial shield generator, you’d better sacrifice some growth for defense. But once the Death Star has blown up and the Empire is swept away, you probably focus more on growth (until a new Sith lord arrives).
This guns vs. butter debate plays out at the cellular level too when it comes to protecting DNA from mutations. If cells expend too much energy to protect their DNA they sacrifice growth, but if they spend too little, they develop too may harmful mutations to survive. And just like with countries, how much protection a cell’s DNA needs depends on its environment.
If cells need to adapt quickly to a changing environment, a high rate of mutation is favored. These cells are more likely to develop a mutation that gains them an advantage over their slower mutating brethren.
A new study by Herr and coworkers in the latest issue of GENETICS calculates the upper limit of the rate of mutation in a diploid yeast. In other words, they figure out how little “spending” on defense these yeast can get away with and survive.
They find that diploid yeast can deal with a 10-fold higher rate of mutation as compared to haploid yeast. This makes sense, since the extra gene copy afforded by being diploid can mask a recessive lethal mutation, but this study is the first to give this idea hard numbers.
The authors had previously generated a number of mutations in POL3, the yeast gene for DNA polymerase δ, that affect its ability to find and/or fix any mistakes made during DNA replication. The study first focused on two mutations affecting accuracy, pol3-L612G and pol3-L612M, and one mutation affecting proofreading, pol3-01. The accuracy mutations caused about a 10-fold increase in the mutation rate, while the proofreading mutation caused anywhere from a 20-100-fold increase. Neither was enough to seriously affect a diploid’s growth.
The next step was to combine accuracy and proofreading mutations into the same gene to figure out if the combination resulted in a higher mutation rate. The authors suspected that it did when they discovered that even though the heterozygotes were fine, their spores were inviable. The POL3/pol3-01,L212M and POL3/pol3-01,L212G strains sporulated just fine, but none of the spores could germinate and grow.
One way to explain this was that the double mutation increased the error rate to the point that it would kill off haploids but not diploids. By looking at mutations in the hemizygous CAN1 gene they could see that the mutation rate in these diploids was indeed at around the haploid threshold. In terms of the CAN1 gene, this mutation rate was around 1X10-3 can1 mutations/cell division.
They next determined the mutation rate by sequencing the genomes of each mutant as well as the wild type. They found a single T-G mutation in the wild type, 1535 point mutations in POL3/pol3-01,L212M and 1003 mutations in POL3/pol3-01,L212G. From this they calculated a mutation rate of around 3-4X10-6/base pair/generation.
Even though this level of mutation kills haploids but not diploids, this does not mean the diploids escaped unscathed. When the heterozygous diploid colonies were subcloned the resulting colonies were variable in size, indicating that their higher mutation rate was catching up with them. This high mutation rate was making them sick.
Given this result, it wasn’t surprising that diploid homozygotes of each double mutant could not survive—the mutation rate was now too high. The strains homozygous for pol3-01,L212M managed to get to around 1000 cells before petering out. Strains homozygous for pol3-01,L212G did even worse—they only made it to around 10 cells.
In a final set of experiments Herr and coworkers used a variety of other mutations to tweak these mutation rates to find the threshold at which diploids fail to survive. Some of these mutations were in POL3 while others were deletions of the MSH2 and/or DUN1 genes. After testing many different combinations, they found that these yeast did pretty well up to around 1X10-3 can1 mutations/cell division (the haploid threshold rate). Then, from 1X10-3 to 1X10-2 can1 mutations/cell division there began a rapid drop off with little to no growth at the end.
So as might be expected, diploids can deal with a significantly higher mutation rate than can haploids. But even though they can, wild type yeast in the lab still have a very low mutation rate. It is like they are living near the Imperial city planet of Coruscant. They are willing to expend the energy to keep their DNA protected.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: DNA replication , mutation , Saccharomyces cerevisiae