New & Noteworthy

Multi-invasion recombination leads to chromothripsis-like phenomenon

August 18, 2023

Homologous recombination is a universal pathway to repair DNA double-stranded breaks, ssDNA gaps, and stalled or collapsed replication forks. Homologous recombination relies on an intact dsDNA donor that is identical to the broken molecule to template the repair event in a copy/paste-type reaction. Typically, this donor is the allelic region on either the sister chromatid or homolog. But what happens when DNA damage falls within, or proximal to, repetitive DNA? A study recently published in Genes and Development by Diedre Reitz and colleagues shows that, in rare cases, homologous recombination can engage two or more repetitive elements in a process termed multi-invasion recombination. 

Using genome-wide sequencing, Reitz and colleagues found that multi-invasion recombination can lead to a cascade of recombination-induced rearrangements, aneuploidies, and secondary DNA breaks. By creating a specialized reporter assay, they discovered two pathways in which multi-invasion intermediates can be resolved into products, thereby forming multi-invasion-induced rearrangements. The MIR1 pathway can occur in any sequence context, generates secondary DSBs, and frequently leads to additional genome rearrangements. The MIR2 pathway occurs only when the recombining donors exhibit substantial homology, and results in an insertion without additional DNA breaks. To better understand the molecular determinants of MIR1, the authors developed a highly sensitive proximity ligation-based assay to detect rare MIR1 translocation events. Using this assay, the authors found that, in contrast to normal repair, MIR1 does not require displacement DNA synthesis to fill in the nucleotides lost due to DNA damage.

These vast rearrangements originating from a single DNA break and resolution via the MIR1 pathway are reminiscent of chromothripsis, a mutational signature characterized by large clustered genomic rearrangements frequently associated with certain cancers. Overall, the findings by Reitz and colleagues provide new mechanistic insight into how complex recombination-dependent rearrangements can occur. 

— Text from Diedre F. Reitz, with edits and link outs from SGD.

Categories: Research Spotlight

Tags: cancer, homologous recombination

A Revelation in Loosening Chromatin to Allow Replication

May 20, 2022

The orderly replication of chromosomes is a daily miracle, driving growth of all higher organisms. Yet chromosomes are packaged into tight little nucleosome balls that must be systematically unpacked before replication can commence. Much is yet to be learned about this process, but a recent study by Chacin et al. in Nature Communications has provided a new piece of the puzzle.

The ATPase Yta7p was originally identified as an element that marked boundaries within chromatin, thereby influencing gene transcription by delineating active versus repressed regions. The protein has domains characteristic of the type-II family of AAA⁺–ATPases. It was previously noted that Yta7p becomes phosphorylated during S phase and that phosphorylation caused eviction of Yta7p from chromatin. However, the reason a barrier protein would need to be specifically modified during S phase remained elusive.

In this new study, Chacin et al. observed replication defects in yta7 mutants and hypothesized that Yta7p might play a heretofore-unknown role in DNA synthesis. To address this hypothesis, the authors purified the protein and demonstrated a hexameric structure similar to known segregases of the AAA⁺-ATPase family. Next, the authors established an in vitro assay to assess the activity of the purified protein on packaged nucleosomes. By means of impressive reconstitution experiments, they showed that Yta7p is recruited to acetylated chromatin but does not have activity on chromatin until Yta7p is specifically activated by phosphorylation.

This activating phosphorylation, they show, is performed by the S-CDK complex (CLB5-CDC28 kinase complex) specifically during S phase. Further, phosphorylation was then shown to stimulate the ATPase function of the Yta7p enzyme.

Using a similar set of reconstitution experiments, the authors then asked questions about the activity of activated Yta7p on naked DNA versus chromatin. They showed that Yta7p did not have an effect on either naked DNA or nonacetylated chromatin, but strongly stimulated active replication on acetylated nucleosomes. From this they propose a model whereby active Yta7p causes nucleosomes to disassemble so that origins of replication are accessible to the replication machinery.

Intriguingly, the YTA7 gene is conserved among eukaryotes, with the human homolog ATAD2 identified as an oncogene overexpressed in assorted cancers. The use of yeast to tease out the role of Yta7p in unpackaging nucleosomes ahead of DNA replication sheds light on the possible role of human ATAD2 in tumorigenesis. The simplicity of yeast once again affords insight into complicated systems within cell biology.

Categories: Research Spotlight

Tags: cancer, chromsome replication, DNA replication, Saccharomyces cerevisiae, yeast model for human disease

Guardians of (the) Chromosome Stability

March 18, 2022

Chromosome instability (CIN), recognized as a hallmark of several cancers, results from error in chromosome segregation leading to differences in both chromosome structure and numbers. Chromosome Segregation protein (CSE4) in budding yeast and CENP-A in humans are examples of an evolutionary conserved histone H3 variant in all eukaryotic centromeres which have a crucial role in efficient chromosome segregation. As such, overexpression of CSE4 (or CENP-A) are known to cause mislocalization of the protein to non-centromeric chromatin leading to CIN.

In an interesting new study in Nucleic Acids Research, Ohkuni et al. have shown how mislocalized Cse4p is removed and targeted for proteasomal degradation, thus preventing CIN. The authors show that Cdc48p-Npl4p-Ufd1p AAA ATPase complex recognizes the mislocalized and polyubiquinated Cse4p and facilitates its removal from the non-centromeric chromatin, targeting it for degradation. The authors demonstrate that the Cdc48p complex targets specifically the mislocalized chromatin-bound Cse4p and not the centromeric Cse4p. Another essential factor involved in this mechanism is an E3 ubiquitin ligase, Psh1p, which polyubiquinates Cse4p and promotes its recognition by Cdc48p-Npl4p-Ufd1p AAA ATPase complex via its cofactor Npl4p.

This paper demonstrates an important role for the Cdc48-Npl4-Ufd1 AAA ATPase complex in removing mislocalized Cse4p from non-centromeric chromatin. It infers that accumulation of mislocalized CENP-A may contribute to aneuploidy in human cancers, thus revealing another pathway to target for treating human diseases.

Categories: Research Spotlight

Tags: cancer, chromosome instability, Protein Mislocalization, Saccharomyces cerevisiae

C-Circles, an ALT Plan for Telomere Restoration

February 18, 2022

The telomerase ribonucleoprotein complex is the primary means by which yeast cells maintain telomeres. However, it turns out that cells lacking functional telomerase have a backup plan to restore telomere length by “alternative lengthening of telomeres” (ALT). ALT employs recombination via extrachromosomal telomere elements called C-circles. In a process for which the reasons remain unclear, C-circles get paired with eroded telomeres at the nuclear pore complex on the nuclear membrane. This pairing requires the SAGA/TREX2 complex and, once paired, the recombination between C-circles and telomeres appears to be effected by Rad59p, the paralog of Rad52p.

This interesting model is described in a recent paper in The EMBO Journal, in which Aguilera et al. adapt a method developed in human cancer studies to detect ALT and C-circles in yeast. In humans, ~10% of cancers depend on ALT for unchecked growth. In yeast, cells with ALT were able to be detected as survivors among telomerase mutant (est2∆) cells.

As other types of extrachromosomal DNA circles were previously reported to associate with the nuclear pore complex, the authors addressed the possibility that C-circles bind the NPC and demonstrated it clearly. They also showed the circles interact with the SAGA/TREX2 complex, which favors telomere recombination.

The novel finding that ALT in yeast so closely mirrors that of some human cancer cells is a boon to study of these cancers. The ability to develop ALT inhibitors in yeast would provide a new set of potential anticancer therapies, making this an ideal model system.

Categories: Research Spotlight

Tags: cancer, cell aging, Saccharomyces cerevisiae, senescence, telomeres, yeast model for human disease

Too Much of a Good Thing Can Be Hazardous to Your Health

July 02, 2018

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When someone says they can “read you like a book”, they probably aren’t saying that they know your entire genome sequence. (…or for you Westworld fans, you certainly hope they aren’t saying they’ve got access to the Delos Incorporated “library”!).

But in fact everyone’s genome CAN be thought of as a book, sort of like a giant cookbook. Within your genome are many “recipes” for gene products — DNA sequences that each give instructions on how, when, and where to make a protein or RNA molecule. The recipes of the genome are used to “cook up” a person!

When a parent cell divides and creates another cell, it makes a copy of its genome “cookbook” and passes it down to the new cell. It’s extremely important to make sure that there aren’t any errors in the newly copied text, as mistakes in recipes for crucial enzymes can have disastrous results for the cell. Indeed, our cells (as well as yeast cells, and in fact, all organisms) have enzymes that are dedicated to scrupulously inspecting the new copies of our genome cookbooks that are made during cell division, and then helping to correct any errors.

A certain class of these enzymes are known as DNA mismatch repair (MMR) proteins. These enzymes carefully proofread the newly made copy of the genome, and if something doesn’t match the original version, they will help fix the error.

You might think that having more of these MMR proteins would be a good thing, because there would be more thorough checking and repairing of the new copy of the genome. But the situation isn’t so simple, especially when it comes to human cancer.

Previous studies show that some cancers and cancer predisposition syndromes have less of the MMR proteins. This makes sense because cancers almost always arise due to genome mutations, and it is known that if a cell has less of the error-checking MMR proteins, there are more genome mutations. However, other studies report a puzzling discrepancy: in some cancers, there are in fact MORE of the MMR proteins rather than less!

In their recent GENETICS study, Chakraborty and coworkers decided to investigate this discrepancy further. They analyzed data from two databases of human cancer genome information, The Cancer Genome Atlas (TCGA) and the cBioPortal for Cancer Genomics, specifically looking at how much the genes encoding MMR proteins were turned on or off in cancer.

They observed that many types of human cancer cells make more of two particular MMR proteins: MSH2 and MSH6. It turns out (as it often does!) that our good buddy Saccharomyces cerevisiae has MMR proteins very similar to the human ones, also encoded by genes called MSH2 and MSH6. So Chakraborty and coworkers made yeast cells that overexpress the yeast genes MSH2 and MSH6 and used the awesome power of yeast genetics (and genomics) (#APOYG!) to investigate whether this might lead to cellular and genome changes like those seen in human cancers.

Using various yeast genetic assays to measure rates of genome alterations such as homologous recombination, mutations, and loss of large chromosome regions, Chakraborty et al. observed increased rates for all of these measures of genome instability in cells when both MSH2 and MSH6 were overexpressed, but not for overexpression of either one alone (or of other MMR proteins). So even though there are more of the “good guy” MMR proteins in these cells, this actually ends up making the genome of the cell MORE likely to get damaging mutations of the same types seen in cancer cells.

So why is too much of a good thing such a bad thing in this case? The authors hypothesize that both Msh2p and Msh6p act together as a joined pair to go to the spot where the chromosomal DNA is actively being copied (“replicated”). When they are both overexpressed, too many of the Msh2p-Msh6p pairs can go to the replication spot and actually interfere with the copying process.

It’s as if you were a medieval scribe carefully copying an illuminated manuscript of a genome cookbook, and instead of one supervisor occasionally checking your work, there are a bunch of people constantly looking over your shoulder and maybe even bumping into your arm, causing you to make mistakes in your writing. They may even make you drop the book you’re copying, scattering pages so that you might leave some out or put them back in the wrong order, seriously messing up your work!

Here’s hoping our cells don’t overdo it with their MMR proteins, so that they can be careful with their cookbook copying job and do it “write”!

by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: cancer, DNA mismatch repair, DNA replication, MSH2, MSH6

Yeast Reveals a Vicious Circle Between Sugar and Cancer

November 10, 2017

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The housing crisis that started in 2006 is a classic example of a vicious circle. As prices went down, some people couldn’t afford their mortgages and so were foreclosed upon. These foreclosed houses entered the market and caused prices to drop further which caused more foreclosures and so on. Only a Herculean effort by the government and the Federal Reserve was able to break this cycle.

Vicious circles can happen in biology too.

In a new study in Nature Communications, Peeters and coworkers have used our friend Saccharomyces cerevisiae to uncover one of these in cancer cells. And it turns out that this yeast is the perfect model organism for this study. (Funny how often that is true…)

Both S. cerevisiae and cancer tend to utilize their sugar through fermentation instead of respiration. This process in yeast is obviously great news for beer and wine drinkers. However, it is not such great news for people with cancer.

In the Warburg effect (named after the scientist who discovered it), the more aggressive a cancer is, the more sugar it ferments. This new study suggests that the consumption of sugar via fermentation and the aggressiveness of the cancer are related in a vicious circle.

Fermentation creates the byproduct fructose-1,6-bisphosphate (Fru1,6bisP) which, according to this study, activates the oncogene Ras which causes the cell to grow faster. As it grows faster, it ferments sugar faster creating more Fru1,6bisP which activates more Ras and so on. The cancer cells grow out of control until doctors apply some Herculean treatment to put a stop to it.

While yeast and cancer cells have a lot in common, wild type yeast isn’t quite up to cancer’s standards when it comes to cancer’s unbridled intake of glucose into the glycolysis pathway. These authors turned yeast a bit more cancer-like in this regard by deleting the TPS1 gene. Tps1p is a yeast hexokinase that tightly regulates yeast’s glucose intake into glycolysis.

Yeast cells without TPS1 deal poorly with glucose and need to be grown in galactose. When Peeters and coworkers added glucose to tps1Δ cells that had been previously grown in galactose, the cells activated Ras, a potent oncogene. This activation caused the cell to undergo apoptosis as assayed by cytochrome c release from the mitochondria, exposure of phosphadityl serine on the plasma membrane and generation of reactive oxygen species.

Deletion of hexokinase 2 eliminated this activation of Ras and suppressed the apoptosis. This suggests that perhaps some buildup of an intermediate metabolite in the glycolytic pathway might be to blame for the Ras activation.

The authors tested this hypothesis by removing the cell wall of wild type yeast cells and adding glycolysis metabolites to the resulting spheroplasts. They found that one metabolite, Fru1,6bisP, activated Ras. Supporting this finding, they found that deletion of both PFK1 and PFK2, two genes that encode phosphofructokinase 1, the enzyme responsible for making Fru1,6bisP, eliminated Ras activation in tps1Δ cells. Together, these data support the idea that Fru1,6bisP is the culprit behind Ras activation in yeast.

All well and good, but what about human cancer cells? Is something similar going on there? You have probably guessed that the answer is yes.

The authors first depleted the level of Fru1,6bisP in two different cell lines by starving them of glucose for 48 hours. They then added back glucose, which has been shown to increase the levels of Fru1,6bisP in cells, and measured the activation level of Ras and two of its downstream targets, MEK and ERK. All three were transiently activated in both HEK293T and Hela Kyoto cell lines. Looks like Fru1,6bisP activates Ras in cancer cells as well.

So this might explain the Warburg effect that I mentioned earlier. The more glucose a cancer cell uses during fermentation, the more Fru1,6bisP it generates. And the more Fru1,6bisP that is made, the more Ras gets activated prompting the cell to grow faster. Which of course results in more glucose getting fermented and so on.

I don’t have time to go into the work these authors did to try to uncover the mechanism by which Fru1,6bisP activates Ras, but suffice it to say that it does not appear to be a direct interaction with Ras. Instead, it appears to work at least partially by disrupting the interaction between Ras and one of its guanine nucleotide exchange factors, Cdc25p (or Sos1 in humans).

And the story is not yet complete. There is still a lot of work to do in pinning down the specifics of this vicious circle. Yeast will undoubtedly be instrumental in helping us work through the rest of the details.

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

Categories: Research Spotlight

Tags: cancer, fermentation, PFK1, PFK2, TPS1, Warburg Effect

Knock out YME1, Luke

April 20, 2017

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In the original Star Wars, Luke destroys the Death Star with a precise strike of proton torpedoes down a small thermal exhaust port. For him it was as easy as bullseyeing “womp rats in my T-16 back home.”

Luke and the rest of the Rebel Alliance learned of this engineered fatal flaw from Jyn and her friends in the prequel Rogue One. With this information the Rebel Alliance was able to keep the rebellion alive long enough to finally bring down the Empire by the end of Return of the Jedi.

It turns out that our friend Saccharomyces cerevisiae has taught us about a fatal flaw in mitochondria. Like proton torpedoes in an exhaust port, when the gene YME1 is inactivated, mitochondria become unstable. But instead of bits of Death Star raining down on nearby planets, mitochondrial DNA (mtDNA) is released into the cytoplasm.

Sometimes this mtDNA can end up in the nucleus and find its way into nuclear DNA. And if the conclusions of a new study in Genome Medicine by Srinivasainagendra and coworkers turns out to be right, this numtogenesis (as the authors call this process) can have profound consequences when it happens in people. Their data suggests that it might lead to cancer or possibly cause cancers to spread.

These researchers searched through whole genomes of colon adenocarcinoma patients and found that these cancer cells had 4.2-fold more mtDNA insertions compared to noncancerous cells from the same patient. They also found that patients with more of these insertions tended to do worse (although the sample sizes were too small to say this definitively).

Why is this happening in the cancer cells? What has caused the mitochondria to give up their DNA?

Srinivasainagendra and coworkers turned to previous work that had been done on the YME1 gene in the yeast S. cerevisiae to find one possible reason. YME1 had been shown to be an important suppressor mtDNA migration to the nucleus. Perhaps this was true in mammalian cells as well.

A search through the genomes of cancers suggested that this seemed to be the case. Around 16% of the colorectal tumors they looked at had a mutated YME1L1 gene, the human homologue of YME1. And mutated YME1L1 genes were found in other tumors as well.

They used CRISPR/Cas9 to directly test the effects of knocking out YME1L1 in the breast cancer cell line MCF-7. The knock out cells had a 4-fold increase in the amount mtDNA in the nuclear fraction compared to cells that still had working YME1L1.

As a final experiment, they used a yeast strain, yme1-1, in which YME1 function was inactivated, to show that the human homologue, YME1L1, could suppress the migration of mtDNA to the nucleus.

This yme1-1 strain has a TRP1 gene encoded in the mtDNA instead of the nucleus. Since the gene cannot be read by the mitochondrial transcription machinery, the only way this yeast strain can survive in the absence of tryptophan is if the TRP1 gene moves from the mitochondrion to the nucleus. 

In their experiment, with vector alone, they got around 1000 TRP+ colonies with yme1-1. When they added back yeast YME1, this number dropped to less than 50 compared to the 100 or so they got when they added the human homologue, YME1L1. So YME1L1 can suppress mtDNA migration to the nucleus.  

Given that YME1L1 was mutated in just a subset of the cancers, it is unlikely that it is the only player in the mtDNA these authors found in the nuclei of cancer cells. But it does look like it is one way this can happen.

And it would have been very hard to fish out the human gene without the critical work that had been done in yeast previously. Yeast shows us the way again. #APOYG

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

Categories: Research Spotlight

Tags: cancer, colon cancer, CRISPR/Cas9, MCF-7, mtDNA, nuMt, nuMtogenesis, YME1, YME1L1

Feed a Cold, Starve a Cancer?

March 02, 2017

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You may have heard the old wives’ tale of feed a cold, starve a fever. Turns out that this isn’t particularly good advice (although some studies do suggest that with a fever, you shouldn’t force-feed yourself). It also turns out to have probably originated in the 19th century and not from Chaucer in the 14th as many websites claim.

But while starving a fever is probably never a good idea, starving a cancer can be. Not by following the medical myth that since cancers use a lot of sugar, you can starve them by cutting down on sugar in your diet. Instead you can starve some cancers by denying them the amino acid asparagine (Asn).

On their way to becoming cancerous, acute lymphoblastic leukemia (ALL) cells lose their ability to make Asn. This means that unlike the cells around it, they need to pull Asn from the blood to make their proteins and to survive.

Doctors exploit this weakness by injecting L-asparaginase amidohydralase (L-ASNase) into patients which starves the cancer cell by depleting Asn levels in the blood. The cells around the cancer cells are fine because they can still make Asn.

Right now doctors use L-ASNase from two different bacterial sources: Escherichia coli and Erwinia chrysanthemi. But if a recent study by Costa and coworkers in Scientific Reports holds up, they might want to think about switching to using the Saccharomyces cerevisiae L-ASNase encoded by the ASP1 gene.

An older study had suggested that the yeast enzyme might be too weak to be useful. This new study finds that this is not the case.

The difference between the older study and this one was the purification protocol. The older study purified the native enzyme through multiple chromatography steps while this study used a single affinity chromatography step. The purified yeast and E. coli versions have comparable activity in this study.

They are also comparable in terms of being able to work with very low concentrations of Asn. This is important as Asn levels are very low in the blood.

What makes the yeast enzyme potentially better is that it is much worse at hydrolyzing a second amino acid, glutamine, than are the bacterial versions. This higher specificity for Asn is important because one of the major side effects of the current treatment is neurotoxicity caused by decreased levels of glutamine in the blood. Since the yeast version hydrolyzes glutamine at a lower rate, they predict patients may not suffer as badly from this side effect with the yeast version.

Of course this is all for naught if the yeast enzyme can’t kill cancer cells! Or if it kills cells indiscriminately.

The S. cerevisiae version was nearly as good as the E.coli version in tissue culture. After 72 hours of incubation, both versions had little effect on normal cells (HUVEC), and both were cytotoxic to the L-ASNase-sensitive cell line MOLT-4 with the E. coli version killing 95% of MOLT-4 cells and the yeast version killing 85% of them.

Taken together these results suggest that the S. cerevisiae version may be an alternative to the bacterial versions. It may be able to kill cancer cells with fewer side effects.

But the yeast version is not the only alternative in town. Another group is engineering the E. coli version to lessen its propensity for hydrolyzing glutamine. Either way it looks like certain leukemia patients may be getting an effective cancer treatment with fewer side effects.

Beer, wine, bread, chocolate, and now maybe a treatment for a nasty form of leukemia. Yeast may be humanity’s best friend. #APOYG!

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: Acute lymphocytic leukemia, cancer, Proteins, Recombinant protein therapy

Too Much of a Good Thing

November 09, 2016

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Like a ruined cookie with too much salt, a cell can go haywire when it has too many copies of certain genes. And of course, cells can deal perfectly well with too many copies of other genes. Just like adding too many chocolate chips to your cookies might make an even better cookie!

Finding out which genes are like salt and which ones are like chocolate chips is of more than just general biological interest. It might help us to explain why cancer happens and to possibly find better treatments.

As you probably know, cancer cells are pretty messed up genetically. Their DNA is littered with mutations, rearrangements and somatic copy number amplifications (SCNAs).

A big reason for this genetic jumble is early DNA changes that increase the rate of mutations in a cell. This “mutator” trait makes a cell more likely to stumble on the mutations it needs to grow out of control or refuse to die.

In their new study in GENETICS, Ang and coworkers set out to find genes that can cause a mutator phenotype when they are part of a SCNA. In other words, which genes lead to an increased mutation rate when expressed at a higher level.

This is important because there are so many SCNAs in a typical cancer cell that it can be hard to figure out which ones matter and which ones don’t (or to put it into cancer parlance, to tell the drivers from the passengers). And despite all of the CRISPR hoopla and other mammalian resources, it would still be a very long process to find “dosage mutator” genes in cell culture and/or living animals.

Which is why Ang and coworkers used our favorite workhorse, the yeast Saccharomyces cerevisiae, to find genes that may cause an increased mutation rate when overexpressed.

The assay is conceptually simple. Yeast that have a functioning CAN1 gene do not survive in the presence of the drug canavanine. So these researchers looked for cells that did better in the presence canavanine when overexpressing a single gene. Presumably, they are surviving because that extra gene resulted in the CAN1 gene being mutated more often because of an increased mutation rate.

They found 37 genes that fit the bill, 18 of which that were involved in biological pathways known to affect genome stability. Combining this with previous studies that looked at gene deletions, this brings the grand total of suspected yeast mutator genes to 210.

Most of these 210 were identified because of mutations that made them stop working which can make figuring out why they cause the mutator phenotype relatively simple. For example, if a mutation kills a gene responsible for fixing DNA mistakes, then you are going to get more DNA mistakes in that cell. It is a little trickier to understand how extra copies of a gene might cause an increased mutation rate.

Ang and coworkers focused on trying to figure out the mechanism behind their top 5 dosage mutator genes: PIF1, MPH1, UBP12, RRM3, and DNA2. Since 4/5 of these code for helicases, they first checked to see if just being a helicase is enough to be a dosage mutator gene. It isn’t.

They retested 48 DNA helicases in their assay and found that none of them caused an increased mutation rate when mutated. There is more to a dosage mutator than being a helicase!

In the next set of experiments, they wanted to determine if the five strains, each overexpressing one of these five genes, had a higher mutation rate by the same mechanism. They tested this by determining the sensitivity of these 5 strains to 3 different DNA damaging agents. The idea is that if they share the same mechanism, they should have the same sensitivity profiles to each of these agents. They did not.

For example, overexpressing MPH1 resulted in a higher sensitivity to all three agents while overexpressing UBP12 only increased sensitivity to two of them. So each strain probably has an increased mutation rate for a different reason.

They next wanted to see if the increased mutation rate was due to a loss or gain of function. They did this by comparing the profiles of strains either deleted for or overexpressing the dosage mutator genes. The idea is that if overexpression leads to a loss of function, then deleting and overexpressing the genes should have the same profile. The three they could test like this did not.

The authors conclude from this that the increased mutation rate for MPH1, UBP12, and RRM3 is most likely due to the gain of an inappropriate function as opposed to a loss of function. In a final set of experiments, Ang and coworkers focused on what that new function might be in their strongest mutant, MPH1.

First they showed that of the three activities associated with Mph1p, only DNA binding and not its ATPase or helicase activities were important for it causing an increased mutation rate when overexpressed. From this they reasoned that perhaps Mph1p was displacing some other important DNA binding protein and that it was this displacement that was causing the increased mutation rate.

Through a set of experiments we don’t have time to go into here, they provided evidence that Mph1p was outcompeting the flap endonuclease Rad27p for DNA binding. This makes some sense as previous work had shown that deleting RAD27 causes mutation rates to go way up. So too much Mph1p keeps Rad27p from getting to where it needs to be with the end result being an increased mutation rate.

All this MPH1 work may have important implications in some human cancers. Nonsense or missense mutations in FANCM, the human homolog of MPH1, are known to make people more likely to get cancer. And there are examples of cancers where FANCM is overexpressed. Perhaps that overexpression results in an increased mutation rate in these cancers.

Yet again yeast is giving researchers new targets for, and new ways to think about, human disease. Thanks, yeast, for finding all of these mutator genes for us to investigate further! #APOYG!

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: cancer, forward mutation, genome-wide, MPH1, mutator, overexpression

Lessons from Yeast: Poisoning Cancer

April 06, 2016

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In the book Dune, the mentat Thufir Hawat is captured by the evil Harkonnens and given a residual poison. He can only stay alive by getting a constant dose of the antidote. Once it is withdrawn, he will die.

A new study in the journal GENETICS by Dodgson and coworkers shows that the same sort of thing can happen to yeast that carry an extra chromosome. In this case, certain genes on the extra chromosome turn out to be like the residual poison. And a second gene turns out to be the antidote.

Once that second gene is deleted, the yeast cell dies. It has been deprived of its antidote.

This synthetic lethal phenotype isn’t just a cool finding in yeast either. Cancer cells invariably have extra and missing chromosomes. If scientists could find similar “antidote genes” in specific types of cancers and target them, then the cancer cell would die. And this would happen without damaging the other cells of the body that have a typical number of chromosomes.

The first thing these researchers did was to make separate yeast strains each with an extra chromosome I, V, VIII, IX, XI, XII, or XVI. The next step was to see what happens when every gene was deleted individually, one at a time, from each strain.

As expected, these yeast did pretty well when a gene on the extra chromosome was deleted. So, for example, a strain with an extra chromosome I tolerated a gene deleted from chromosome I. This makes sense as this just brings that gene back to its normal copy number.

But this was not the case with chromosomes VIII and XI. Here deleting genes on the extra chromosome often had a negative effect. This suggested that the screen probably had a high number of false positives and these researchers later confirmed this.

Likely reasons for the high number of false positives include the strain with the extra chromosome being W303 and the deletion strain being S288C, errors in the deletion collection itself, and what they refer to as neighboring gene effects. Basically this last one is the effect that deleting a gene has on nearby genes.

Once Dodgson and coworkers corrected for these problems, they found two broad sets of phenotypes – general and chromosome specific.

The general ones were the ones shared by most or all of the strains. These were deletions that affected the yeast no matter which chromosome they had an extra copy of.

For the most part, these genes were enriched for the Gene Ontology (GO) term vesicle-mediated transport, indicating that they have something to do with the transportation of substances in membrane-bounded vesicles. For example, deletion of MNN10, HOC1, and MNN11, genes all involved in protein transport and membrane-related processes, had a negative effect on many of the yeast strains with an extra chromosome. Consistent with this, brefeldin A, a drug that targets protein trafficking, negatively affected most of the strains.

Another gene that affected many of these strains when deleted was TPS1. This gene encodes a subunit of trehalose-6-phosphate synthase, a key enzyme for making trehalose, a molecule that helps yeast deal with stress. Perhaps not surprisingly, having an extra chromosome is stressful!

In addition to the genes that affect many strains with an extra chromosome, there were also genes that were chromosome specific. The best characterized of these was the EDE1 gene in the strain with an extra chromosome IX. Deleting EDE1 in this strain increased its doubling time by more than 80 minutes while only causing an increase of 5 minutes in the doubling time of wild type yeast. This was a severe phenotype in their assay.

They next tried to find which gene on chromosome IX might be responsible for the severe effect of deleting EDE1. Since EDE1 is known to be involved in endocytosis, they looked for genes involved in the same process. And they found one – PRK1.

The strain with a deleted EDE1 gene and an extra chromosome IX was rescued by deleting one copy of the PRK1 gene. The extra PRK1 gene was the poison and the EDE1 gene was the antidote.

If a similar pair could be found in cancers that often have the same set of extra chromosomes, then perhaps scientists could develop drugs that target an antidote gene. Now the cancer cells would die and the “normal” cells would be fine. Thanks again, yeast, for pointing us toward new ways to treat human disease.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: aneuploidy, cancer, synthetic lethal

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