New & Noteworthy

The Baby Bear of Proteins

July 31, 2013

In the story of Goldilocks and the Three Bears, Goldilocks always likes Baby Bear’s stuff best. Baby Bear has the most comfortable bed, the best porridge, and so on.

The reason Goldilocks likes Baby Bear’s things are that they are just right. They are neither too hard nor too soft, too hot nor too cold, too big nor too little.

Turns out that when it comes to certain proteins, the yeast S. cerevisiae is sort of like Goldilocks…it likes to have them at just the right levels. Too much or too little protein can throw things out of whack.

This idea is supported in a new study in GENETICS, where Sasanuma and coworkers find that a key helicase in yeast, Srs2p, needs to be present in just the right amounts for meiotic recombination to go off without a hitch. In particular, they show that this protein affects meiotic recombination by interfering with the assembly of filaments containing another protein, Rad51p.

Meiotic recombination starts off with Spo11p making a double stranded (DS) break in the DNA. This DS DNA is then trimmed back so that there is a 3′ overhang of single stranded DNA which is then coated with replication protein A (RPA), Rad51p, and Dmc1p. The coated single stranded DNA then invades a stretch of homologous DNA and recombination can begin. 

One of the first things Sasanuma and coworkers did was to show that toying with Srs2p levels has a negative effect on meiosis in general. Too little Srs2p brings spore viability down to 36.8% of wild type, and overexpressing it brings spore viability down to 22.4%.  Clearly Srs2p is a bit like Goldilocks…the amount has to be just right.

The authors next set out to determine how overexpressing Srs2p affects meiosis so profoundly. They showed that too much Srs2p delays the start of meiosis, causes chromosomes to end up in the wrong places, and stunts the repair of DS DNA breaks. Basically, extra Srs2p inhibits meiotic recombination.

They next looked at areas on the DNA where both Rad51p AND Dcm1p were bound, and found that too much Srs2p keeps Rad51p but not Dcm1p off the DNA. When either of these proteins binds to DNA, it forms foci that are visible as dots when the proteins are detected with fluorescent antibodies. While a wild type strain had roughly equal numbers of Dcm1p and Rad51p foci, there were four fold fewer Rad51p foci when Srs2p was overexpressed. Clearly Srs2p was keeping Rad51p-DNA complexes from forming. 

Srs2p can act as a translocase, and it can also bind Rad51p. Sasanuma and coworkers asked which of these functions is essential to its ability to disassemble Rad51p filaments on DNA. Using srs2 mutants that were blocked in just one of these functions, they showed that the translocase mutant was completely unable to remove Rad51p from DNA during meiosis. The Rad51p-binding mutant could still cause Rad51p to dissociate from chromosomes, although at a reduced rate compared to wild type. So the translocase activity is essential, while Rad51p binding is not.

Although it was known that in vitro Srs2p can cause Rad51p-DNA filaments to disassemble, this study is the first to establish that it actually happens in vivo during meiosis. The requirement for the translocase activity suggests that Srs2p may actually move along the filaments as it disassembles them. And this work also shows that just like Goldilocks with her bowl of porridge, the cell needs an amount of Srs2p that is not too big, not too little, but just right.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Size Isn’t All That Matters

July 23, 2013

Sometimes the pressures and stresses of everyday life can make some people go a little crazy…they lose a few of their marbles.  The same thing can happen to a cell too.  The only difference is that instead of losing their marbles, cells can lose their chromosomes!

There are all sorts of mechanisms in place to make sure that a cell has just the right number of chromosomes.  Still, sometimes all these systems fail and a chromosome is lost.  This can be catastrophic for a cell and, as an important part of cancer, catastrophic for the whole body too.

Given how important having the right number of chromosomes is, it is surprising how little we know about what makes a particular chromosome more likely to fly the coop.  Kumaran and coworkers set out to change this in their new study in PLOS ONE.

First off, they showed that some chromosomes in S. cerevisiae are indeed more likely to be lost than other ones and that there was a surprisingly wide range of stabilities.  For example, chromosomes XIII and XIV were thousands of times more stable than chromosome III.

One key factor in stability was chromosome size—the smaller the chromosome the more likely it was to be lost.  But chromosome III showed that size was not the whole story.  It was five times more likely to be lost than the smallest chromosome.

The authors next set out to determine what about chromosome III made it so flighty.  By creating a hybrid of chromosomes III and IX, they were able to show that there was no single site that made chromosome III so unstable.  They were also able to rule out the idea that HML, HMR, and the MAT locus made chromosome III more likely to be lost.

They next focused on the centromere because it is such an important player in chromosome segregation.  They created a series of plasmids using the centromeres from chromosomes III, IX, XII, XIV, and XV and found that the ones from chromosome III and chromosome XV were around 5-fold less stable than the other chromosomes. While they do not have a good explanation for why chromosome III and XV fared the same in their assay, the result did suggest that at least part of the instability of chromosome III could be explained by its centromere.

As a final experiment, they determined the frequency of chromosome loss in mad2 deletion mutants.  They did this because MAD2 is involved in the spindle checkpoint and so is a key mediator of chromosome stability.  They found that deleting this gene significantly increased the loss of other chromosomes, but chromosome III was 3-6 fold less affected by the loss of MAD2.  It was almost as if the centromere of chromosome III was already somewhat compromised for its interaction with the spindle.

The authors aren’t sure yet how chromosome III got to be so unstable. It could be that random mutations just made its centromere less effective. But another interesting possibility is that it might be under selective pressure. Carrying the mating type loci, chromosome III could be considered to be equivalent to a sex chromosome in larger eukaryotes, and we know that those chromosomes are under different evolutionary constraints from other chromosomes. Maybe S. cerevisiae just can’t take the pressure!

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Regulation Information Integrated into SGD

July 18, 2013

The Locus Summary pages of 147 DNA-binding transcription factors (TFs; retrieve the list) now include a new tabbed page, Regulation. This page contains information on the regulatory targets of the TF, its binding sites, and its domains and motifs, as well as a free-text paragraph summarizing its biological context. Take a look at a brief video, below, that explains the different kinds of data found on the Regulation tab. In addition to viewing these data page by page, you can download them all using SGD’s data search and retrieval tool, YeastMine. Click on “Regulation” in the YeastMine menu bar to view the predefined templates for regulation data searches.

 

New Regulation Data at SGD from yeastgenome on Vimeo.

Oxidation: Maybe Not SO Bad After All

July 11, 2013

You don’t have to be a scientist to get the message that oxidation is bad and antioxidants are good. Just go to the vitamin aisle of your local supermarket, or listen to the ads on late-night TV.  You’ll quickly find out that oxidation caused by free radicals is the reason for aging, and antioxidants are the fountain of youth.  Of course you shouldn’t believe everything you hear…

Things just aren’t that clear when you take a good hard look at aging. Yes, oxidation happens, but there actually isn’t solid experimental proof that it causes aging. In mice, this connection has not panned out at all: lowering the ability to sop up oxidants, by knocking out an antioxidant enzyme, does not shorten the mouse’s life.

In a recent eLife paper, joint first authors Brandes and Tienson and their coworkers used our favorite experimental subject, Saccharomyces cerevisiae, to see if oxidation is a cause or just a consequence of aging. They generated a ton of data about oxidation during aging and did not find any evidence for causation.  Instead they came to the surprising conclusion that the trigger for aging may actually be a sudden drop in the levels of the coenzyme NADPH.

The first step, published previously by this group, was to come up with a very sensitive assay for protein oxidation. The amino acid cysteine can act as a sensor for levels of oxidation, as its sulfur-containing thiol group can be oxidized and reduced. Their technique, known as OxICAT, detects the ratio of reduced to oxidized thiol groups on cysteine residues for individual proteins. They can do this for hundreds of proteins at the same time.

In the current study, they looked at the oxidation state of cysteine residues in about 300 different proteins and also measured the levels of several different metabolites related to the redox state of the cell. All of these data were collected over time in aging yeast cells, both under normal conditions and under conditions simulating caloric restriction or starvation.  These last conditions were included because a lower-calorie diet has been shown to slow down aging, in yeast as well as in animals.

Oxidation of proteins definitely did increase over time. But if oxidation were the cause of cell death, you would expect that it would increase steadily and at some maximum point, the cells would die. Surprisingly, that didn’t happen.

Instead, different groups of proteins were oxidized with different kinetics. The most sensitive proteins (about 10% of the set that they studied) were oxidized 48 hours before the cells started to lose viability. This set included some conserved proteins that are important in maintaining oxidation-reduction balance in the cell, such as the thioredoxin reductase Trr1p. 

But it wasn’t only those especially sensitive proteins that were oxidized. In a second wave of oxidation, almost all the remaining proteins (80%) were oxidized at 24 hours before death. And even with so many proteins oxidized the cells were still metabolically active, with ATP levels near normal. So massive oxidation did not equal instant death for these cells.

As predicted, a low-calorie diet slowed down the whole process. The pattern looked a lot like it did in cells on a normal diet, but there was more time between the waves of oxidation and before the end of viability.

The authors also looked at what happened to different metabolites during aging. One key metabolite is the coenzyme NADPH: it donates electrons to the thioredoxin system that helps balance oxidation and reduction. They found that even before any changes in oxidation are detectable, levels of NADPH decrease very suddenly. The authors speculate that this decrease starts the collapse in redox potential that ends in the death of the cell. The oxidation of protein thiols is an effect rather than a cause, and could actually be a way for the cell to sense its redox state and possibly regulate it. NADPH levels have been seen to decrease in aging rats as well, suggesting that this could be a universal part of the aging process.

The results of this study are too voluminous to describe fully here, but they raise a lot of intriguing questions. Some proteins never got oxidized – what protected them? Are NADPH levels really the trigger for aging, and if so, what causes the sudden decrease? Is oxidation of cysteines actually part of a sensory mechanism? And if that’s true, would preventing oxidation really be such a good thing? This may be another good reason to turn off late-night TV.

by Maria Costanzo, Ph.D., Senior Biocurator, SGD