Research Area: Microbiology

Detangling DNA replication

Researchers identify an essential protein that helps enzymes relax overtwisted DNA so each strand can be copied during cell division.

Raleigh McElvery | Department of Biology
September 18, 2018

DNA is a lengthy molecule — approximately 1,000-fold longer than the cell in which it resides — so it can’t be jammed in haphazardly. Rather, it must be neatly organized so proteins involved in critical processes can access the information contained in its nucleotide bases. Think of the double helix like a pair of shoe laces twisted together, coiled upon themselves again and again to make the molecule even more compact.

However, when it comes time for cell division, this supercoiled nature makes it difficult for proteins involved in DNA replication to access the strands, separate them, and copy them so one DNA molecule can become two.

Replication begins at specific regions of the chromosome where specialized proteins separate the two strands, pulling apart the double helix as you would the two shoe laces. However, this local separation actually tangles the rest of the molecule further, and without intervention creates a buildup of tension, stalling replication. Enter the enzymes known as topoisomerases, which travel ahead of the strands as they are being peeled apart, snipping them, untwisting them, and then rejoining them to relieve the tension that arises from supercoiling.

These topoisomerases are generally thought to be sufficient to allow replication to proceed. However, a team of researchers from MIT and the Duke University School of Medicine suggests the enzymes may require guidance from additional proteins, which recognize the shape characteristic of overtwisted DNA.

“We’ve known for a long time that topoisomerases are necessary for replication, but it’s never been clear if they were sufficient on their own,” says Michael Laub, an MIT professor of biology, Howard Hughes Medical Institute Investigator, and senior author of the study. “This is the first paper to identify a protein in bacteria, or eukaryotes, that is required to localize topoisomerases ahead of replication forks and to help them do what they need to do there.”

Postdoc Monica Guo ’07 and former graduate student Diane Haakonsen PhD ’16 are co-first authors of the study, which appeared online in the journal Cell on Sept. 13.

Necessary but not sufficient

Although it’s well established that topoisomerases are crucial to DNA replication, it has now becoming clear that we know relatively little about the mechanisms regulating their activity, including where and when they act to relieve supercoiling.

These enzymes fall into two groups, type I and type II, depending on how many strands of DNA they cut. The researchers focused on type II topoisomerases found in a common species of freshwater bacteria, Caulobacter crescentus. Type II topoisomerases in bacteria are of particular interest because a number of antibiotics target them in order to prevent DNA replication, treating a wide variety of microbial infections, including tuberculosis. Without topoisomerases, the bacteria can’t grow. Since these bacterial enzymes are unique, poisons directed at them won’t harm human topoisomerases.

For a long time, type II topoisomerases were generally assumed adequate on their own to manage the overtwisted supercoils that arise during replication. Although researchers working in E. coli and other, higher organisms have pinpointed additional proteins that can activate or repress these enzymes, none of these proteins were required for replication.

Such findings hinted that there might be similar interactions taking place in other organisms. In order to understand the protein factors involved in compacting Caulobacter DNA — regulating topoisomerase activity specifically — the researchers screened their bacteria for proteins that bound tightly to supercoiled DNA. From there, they honed in on one protein, GapR, which they observed was essential for DNA replication. In bacteria missing GapR, the DNA became overtwisted, replication slowed, and the bacteria eventually died.

Surprisingly, the researchers found that GapR recognized the structure of overtwisted DNA rather than specific nucleotide sequences.

“The vast majority of DNA-binding proteins localize to specific locations of the genome by recognizing a specific set of bases,” Laub says. “But GapR basically pays no attention to the actual underlying sequence — just the shape of overtwisted DNA, which uniquely arises in front of replication forks and transcription machinery.”

The crystal structure of the protein bound to DNA, solved by Duke’s Maria Schumacher, revealed that GapR recognizes the backbone of DNA (rather than the bases), forming a snug clamp that encircles the overtwisted DNA. However, when the DNA is relaxed in its standard form, it no longer fits inside the clamp. This might signify that GapR sits on DNA only at positions where topoisomerase is needed.

An exciting milestone

Although GapR appears to be required for DNA replication, it’s still not clear precisely how this protein promotes topoisomerase function to relieve supercoiling.

“In the absence of any other proteins, GapR is able to help type II topoisomerases remove positive supercoils faster, but we still don’t quite know how,” Guo says. “One idea is that GapR interacts with topoisomerases, recognizing the overtwisted DNA and recruiting the topoisomerases. Another possibility is that GapR is essentially grabbing onto the DNA and limiting the movement of the positive supercoils, so topoisomerases can target and eliminate them more quickly.”

Anthony Maxwell, a professor of biological chemistry at the John Innes Centre who was not involved with the study, says the buildup of DNA supercoils is a key problem in both bacterial replication and transcription.

“Identifying GapR and its potential role in controlling supercoiling in vivo is an exciting milestone in understanding the control of DNA topology in bacteria,” he says. “Further work will be required to show how exactly these proteins cooperate to maintain bacterial genomic integrity.”

According to Guo, the study provides insight into a fundamental process — DNA replication — and the ways topoisomerases are regulated, which could extend to eukaryotes.

“This was the first demonstration that a topoisomerase activator is required for DNA replication,” she says. “Although there’s no GapR homolog in higher organisms, there could be similar proteins that recognize the shape of the DNA and aid or position topoisomerases.”

This could open up a new field of drug research, she says, targeting activators like GapR to increase the efficacy of existing topoisomerase poisons to treat conditions like respiratory and urinary tract infections. After all, many topoisomerase inhibitors have become less effective due to antibiotic resistance. But only time will tell; there is still much to learn in order to untangle the complex process of DNA replication, along with its many twists and turns.

The research was funded by NIH grants, the HHMI International Predoctoral Fellowship, and the Jane Coffin Childs Memorial Fellowship.

Countering mitochondrial stress

Scientists discover a pathway that monitors a protein import into mitochondria and elicits a cellular response when the process goes awry.

Raleigh McElvery | Department of Biology
April 13, 2018

If there’s one fact that most people retain from elementary biology, it’s that mitochondria are the powerhouses of the cell. As such, they break down molecules and manufacture new ones to generate the fuel necessary for life. But mitochondria rely on a stream of proteins to sustain this energy production. Nearly all their proteins are manufactured in the surrounding gel-like cytoplasm, and must be imported into the mitochondria to keep the powerhouse running.

A duo of MIT biologists has revealed what happens when a traffic jam of proteins at the surface of the mitochondria prevents proper import. They describe how the mitochondria communicate with the rest of the cell to signal a problem, and how the cell responds to protect the mitochondria. This newly-discovered molecular pathway, called mitoCPR, detects import mishaps and preserves mitochondrial function in the midst of such stress.

“This is the first mechanism identified that surveils mitochondrial protein import, and helps mitochondria when they can’t get the proteins they need,” says Angelika Amon, the Kathleen and Curtis Marble Professor of Cancer Research in the MIT Department of Biology, who is also a member of the Koch Institute for Integrative Cancer Research at MIT, a Howard Hughes Medical Institute Investigator, and senior author of the study. “Responses to mitochondrial stress have been established before, but this one specifically targets the surface of the mitochondria, clearing out the misfolded proteins that are stuck in the pores.”

Hilla Weidberg, a postdoc in Amon’s lab, is the lead author of the study, which appears in Science on April 13.

Fueling the powerhouse

Mitochondria likely began as independent entities long ago, before being engulfed by host cells. They eventually gave up control and moved most of their important genes to a different organelle, the nucleus, where the rest of the cell’s genetic blueprint is stored. The protein products from these genes are ultimately made in the cytoplasm outside the nucleus, and then guided to the mitochondria. These “precursor” proteins contain a special molecular zip code that guides them through the channels at the surface of the mitochondria to their respective homes.

The proteins must be unfolded and delicately threaded through the narrow channels in order to enter the mitochondria. This creates a precarious situation; if the demand is too high, or the proteins are folded when they shouldn’t be, a bottleneck forms that none shall pass. This can simply occur when the mitochondria expand to make more of themselves, or in diseases like deafness-dystonia syndrome and Huntington’s.

“The machinery that we’ve identified seems to evict proteins that are sitting on the surface of the mitochondria and sends them for degradation,” Amon says. “Another possibility is that this mitoCPR pathway might actually unfold these proteins, and in doing so give them a second chance to be pushed through the membrane.”

Two other pathways were recently identified in yeast that also respond to accumulated mitochondrial proteins. However, both simply clear protein refuse from the cytoplasm around the mitochondria, rather than removing the proteins collecting on the mitochondria themselves.

“We knew about various responses to mitochondrial stress, but no one had described a response to protein import defects that specifically protected the mitochondria, and that’s exactly what mitoCPR does,” Weidberg says. “We wanted to know how the cell reacts to these problems, so we set out to overload the import machinery, causing many proteins to rush into the mitochondrion at the same time and clog the pores, triggering a cellular response.”

“What makes our cells absolutely dependent on mitochondria is one of those million-dollar questions in cell biology,” says Vlad Denic, professor of molecular and cellular biology at Harvard University. “This study reveals an interesting flip-side to that question: When you make mitochondrial life artificially tough, are they programmed to say ‘help us’ so the host cell comes to their rescue? The possible ramifications of such work in terms of human development and disease could be very impressive.”

A pathway to understanding

Roughly two decades ago, researchers began to notice that the genes required to defend cells against drugs and other foreign substances — together, called the multidrug resistance (MDR) response — were also expressed in yeast mitochondrial mutants for some unknown reason. This suggested that the protein in charge of binding to the DNA and initiating the MDR response must have a dual purpose, sometimes triggering a second, separate pathway as well. But precisely how this second pathway related to mitochondria remained a mystery.

“Twenty years ago, scientists recognized mitoCPR as some kind of mechanism against mitochondrial dysfunction,” Weidberg says. “Today we’ve finally characterized it, given it a name, and identified its precise function: to help mitochondrial protein import.”

As the import process slows, Amon and Weidberg determined that the protein that initiates mitoCPR — the transcription factor Pdr3 — binds to DNA within the nucleus, inducing the expression of a gene known as CIS1. The resultant Cis1 protein binds to the channel at the surface of the mitochondrion, and recruits yet another protein, the AAA+ adenosine triphosphatase Msp1, to help clear unimported proteins from the mitochondrial surface and mediate their degradation. Although the MDR response pathway differs from that of mitoCPR, both rely on Pdr3 activation. In fact, mitoCPR requires it.

“Whether the two pathways interact with one another is a very interesting question,” Amon says. “The mitochondria make a lot of biosynthetic molecules, and blocking that function by messing with protein import could lead to the accumulation of intermediate metabolites. These can be toxic to the cell, so you could imagine that activating the MDR response might pump out harmful intermediates.”

The question of what activates Pdr3 to initiate mitoCPR is still unclear, but Weidberg has some ideas related to signals stemming from the build-up of toxic metabolite intermediates. It’s also yet to be determined whether an analogous pathway exists in more complex organisms, although there is some evidence that the mitochondria do communicate with the nucleus in other eukaryotes besides yeast.

“This was just such a classic study,” Amon says. “There were no sophisticated high-throughput methodologies, just traditional, simple molecular biology and cell biology assays with a few microscopes. It’s almost like something you’d see out of the 1980s. But that just goes to show — to this day — that’s how many discoveries are made.”

The research was funded by the National Institutes of Health and by the Koch Institute Support (core) Grant from the National Cancer Institute. Amon is also an investigator of the Howard Hughes Medical Institute and the Glenn Foundation for Biomedical Research. Weidberg was supported by the Jane Coffin Childs Memorial Fund, the European Molecular Biology Organization Long-Term Fellowship, and the Israel National Postdoctoral Program for Advancing Women in Science.

Scientists find different cell types contain the same enzyme ratios

New discovery suggests that all life may share a common design principle.

Justin Chen | Department of Biology
March 29, 2018

By studying bacteria and yeast, researchers at MIT have discovered that vastly different types of cells still share fundamental similarities, conserved across species and refined over time. More specifically, these cells contain the same proportion of specialized proteins, known as enzymes, which coordinate chemical reactions within the cell.

To grow and divide, cells rely on a unique mixture of enzymes that perform millions of chemical reactions per second. Many enzymes, working in relay, perform a linked series of chemical reactions called a “pathway,” where the products of one chemical reaction are the starting materials for the next. By making many incremental changes to molecules, enzymes in a pathway perform vital functions such as turning nutrients into energy or duplicating DNA.

For decades, scientists wondered whether the relative amounts of enzymes in a pathway were tightly controlled in order to better coordinate their chemical reactions. Now, researchers have demonstrated that cells not only produce precise amounts of enzymes, but that evolutionary pressure selects for a preferred ratio of enzymes. In this way, enzymes behave like ingredients of a cake that must be combined in the correct proportions and all life may share the same enzyme recipe.

“We still don’t know why this combination of enzymes is ideal,” says Gene-Wei Li, assistant professor of biology at MIT, “but this question opens up an entirely new field of biology that we’re calling systems level optimization of pathways. In this discipline, researchers would study how different enzymes and pathways behave within the complex environment of the cell.”

Li is the senior author of the study, which appears online in the journal Cell on March 29, and in print on April 19. The paper’s lead author, Jean-Benoît Lalanne, is a graduate student in the MIT Department of Physics.

An unexpected observation

For more than 100 years, biologists have studied enzymes by watching them catalyze chemical reactions in test tubes, and — more recently — using X-rays to observe their molecular structure.

And yet, despite years of work describing individual proteins in great detail, scientists still don’t understand many of the basic properties of enzymes within the cell. For example, it is not yet possible to predict the optimal amount of enzyme a cell should make to maximize its chance of survival.

The calculation is tricky because the answer depends not only on the specific function of the enzyme, but also how its actions may have a ripple effect on other chemical reactions and enzymes within the cell.

“Even if we know exactly what an enzyme does,” Li says, “we still don’t have a sense for how much of that protein the cell will make. Thinking about biochemical pathways is even more complicated. If we gave biochemists three enzymes in a pathway that, for example, break down sugar into energy, they would probably not know how to mix the proteins at the proper ratios to optimize the reaction.”

The study of the relative amounts of substances — including proteins — is known as “stoichiometry.” To investigate the stoichiometry of enzymes in different types of cells, Li and his colleagues analyzed three different species of bacteria — Escherichia coli, Bacillus subtilis, and Vibrio natriegens — as well as the budding yeast Saccharomyces cerevisiae. Among these cells, scientists compared the amount of enzymes in 21 pathways responsible for a variety of tasks including repairing DNA, constructing fatty acids, and converting sugar to energy. Because these species of yeast and bacteria have evolved to live in different environments and have different cellular structures, such as the presence or lack of a nucleus, researchers were surprised to find that all four cells types had nearly identical enzyme stoichiometry in all pathways examined.

Li’s team followed up their unexpected results by detailing how bacteria achieve a consistent enzyme stoichiometry. Cells control enzyme production by regulating two processes. The first, transcription, converts the information contained in a strand of DNA into many copies of messenger RNA (mRNA). The second, translation, occurs as ribosomes decode the mRNAs to construct proteins. By analyzing transcription across all three bacterial species, Li’s team discovered that the different bacteria produced varying amounts of mRNA encoding for enzymes in a pathway.

Different amounts of mRNA theoretically lead to differences in protein production, but the researchers found instead that the cells adjusted their rates of translation to compensate for changes in transcription. Cells that produced more mRNA slowed their rates of protein synthesis, while cells that produced less mRNA increased the speed of protein synthesis. Thanks to this compensation, the stoichiometry of enzymes remained constant across the different bacteria.

“It is remarkable that E. coli and B. subtilis need the same relative amount of the corresponding proteins, as seen by the compensatory variations in transcription and translation efficiencies,” says Johan Elf, professor of physical biology at Uppsala University in Sweden. “These results raise interesting questions about how enzyme production in different cells have evolved.”

“Examining bacterial gene clusters was really striking,” lead author Lalanne says. “Over a long evolutionary history, these genes have shifted positions, mutated into different sequences, and been bombarded by mobile pieces of DNA that randomly insert themselves into the genome. Despite all this, the bacteria have compensated for these changes by adjusting translation to maintain the stoichiometry of their enzymes. This suggests that evolutionary forces, which we don’t yet understand, have shaped cells to have the same enzyme stoichiometry.”

Searching for the stoichiometry regulating human health

In the future, Li and his colleagues will test whether their findings in bacteria and yeast extend to humans. Because unicellular and multicellular organisms manage energy and nutrients differently, and experience different selection pressures, researchers are not sure what they will discover.

“Perhaps there will be enzymes whose stoichiometry varies, and a smaller subset of enzymes whose levels are more conserved,” Li says. “This would indicate that the human body is sensitive to changes in specific enzymes that could make good drug targets. But we won’t know until we look.”

Beyond the human body, Li and his team believe that it is possible to find simplicity underlying the complex bustle of molecules within all cells. Like other mathematical patterns in nature, such as the the spiral of seashells or the branching pattern of trees, the stoichiometry of enzymes may be a widespread design principle of life.

The research was funded by the National Institutes of Health, Pew Biomedical Scholars Program, Sloan Research Fellowship, Searle Scholars Program, National Sciences and Engineering Research Council of Canada, Howard Hughes Medical Institute, National Science Foundation, Helen Hay Whitney Foundation, Jane Coffin Childs Memorial Fund, and the Smith Family Foundation.

A Vision for Science

Clare Harding's microscope image of Toxoplasma gondii parasites is one of this year's winners at the Koch Institute Image Awards

Nicole Giese Rura
March 9, 2018

Scientists use a variety of approaches to unravel the functions of organisms, cells, and even molecules, and some of these approaches produce images that are as stunning as they are informative.  Since 2011, the annual Koch Institute Image Awards, conducted by The Koch Institute for Integrative Cancer Research at MIT, has honored outstanding images created by life science and biomedical researchers in the MIT community.

This year, one of the winning pictures was created by Clare Harding, a postdoctoral researcher in the lab of Whitehead Institute Member and MIT assistant professor of biology, Sebastian Lourido. Harding and the other winners were lauded last night at a gala opening of the exhibit on the Koch building’s ground floor where the winning images will be on display for the coming year.

Whitehead has participated in this contest since its inception, with winning images by Gianluca De Rienzo (postdoctoral researcher in Whitehead Member Hazel Sive’s lab) in 2011, Rob Mathis (graduate student in Whitehead Member Piyush Gupta’s lab) in 2013, Daphne Superville (undergraduate student in Gupta’s lab) in 2015, Dexter Jin (graduate student in Gupta’s lab) in 2016, and Samuel A. LoCascio and Kutay Deniz Atabay (graduate students in Whitehead Member Peter Reddien’s lab) in 2017.

In Harding’s striking entry this year, each white and blue “petal” of the rosettes is a single-celled Toxoplasma gondii, the parasite that causes toxoplasmosis infection. This image was taken moments before the individual parasites comprising the daisy-like clusters would have triggered a massive, coordinated “egress”, which would destroy the host cell they had called home. The host’s nucleus is the large blue oblong jutting in from the upper left (host and parasite DNA are marked blue), and the red dapple marks a molecule in the host cell’s internal skeleton, called tubulin.

Toxoplasmosis infects about 25% of the world’s population and can cause serious disease in pregnant women, infants, and immunocompromised people. Not only is the Lourido lab’s work on T. gondii revealing important clues about this disease, but their research can also shed light on T. gondii’s close cousins on the evolutionary tree: Plasmodium spp., which cause malaria and contribute to more than a million deaths each year; and Cryptosporidium spp., which cause cryptosporidiosis, a gastrointestinal illness that can be fatal in those with a compromised immune system.

Harding’s research in the Lourido lab is focused on GAPM1a, a structural protein that forms a layer directly under T. gondii’s outer membrane and plays a similar architectural role in Plasmodium. This protein scaffold (marked as white in the image) is so vital that it is one of the first things established within daughter cells, which appear in the image as two small white spheres within some of the larger parasites. Parasites lacking the GAPM1a scaffold degrade into amorphous blobs that are unable to infect new host cells—a visual testament of how important this protein is to the parasites.

Light microscopy images like Harding’s are created by passing or reflecting light off of a sample and using one or more lenses to magnify the resulting representation. According to Wendy Salmon, the light microscopy specialist at Whitehead’s W.M. Keck Biological Imaging Facility and a two-time Koch Image Awards judge, all microscopy-based images are imperfect representations of the samples that they depict, because light microscopy is limited by the physics of the light shined on the sample and the glass that comprises the lenses. To push beyond the boundaries of physics and reveal the otherwise invisible, Harding employed two techniques: fluorescent markers and structured illumination microscopy.

Using a light microscope alone, the GAPM1a protein is indiscernible within T. gondii parasites.  But by genetically modifying the parasite to produce the GAPM1a protein fused to a green fluorescent protein, Harding could shine a particular wavelength of light onto the sample and cause the fluorescent protein to glow, thereby illuminating GAPM1a’s presence.

In addition to being able to identify the protein she is studying in a sample, Harding has the additional challenge that the parasites are so tiny—5 micrometers in length, or about 1/16th the width of a human hair—that they are beyond the resolution of light microscopy. In order to visualize the GAPM1a scaffold, Harding used a technique called structured illumination microscopy, which takes advantage of the properties of light in order to see things half the size of what is visible with a conventional light microscope. In this technique, the microscope casts a grid of light onto the specimen and takes images as the grid rotates. The resulting data from the images are processed using an algorithm that reconstructs the specimen’s appearance, enhancing its resolution.

Harding has been working with T. gondii for more than three years and microscopy has always played a major role in her work, but her appreciation for the science and art of microscopy has recently flourished.

“I like microscopy partly because it’s beautiful and partly because with a lot of other techniques, you need to interpret the data. With microscopy, you know what you’re looking at is right there,” says Harding, who is thrilled to have her work featured in the Koch Institute Public Galleries. “I definitely fell in love with microscopy right away. The first time I did it, I realized how much there is to a cell. Even just staining the DNA in a cell, suddenly you can see stars.”

Scientists deliver high-resolution glimpse of enzyme structure

New finding suggests differences in how humans and bacteria control production of DNA’s building blocks.

Anne Trafton | MIT News Office
February 20, 2018

Using a state-of-the-art type of electron microscopy, an MIT-led team has discovered the structure of an enzyme that is crucial for maintaining an adequate supply of DNA building blocks in human cells.

Their new structure also reveals the likely mechanism for how cells regulate the enzyme, known as ribonucleotide reductase (RNR). Significantly, the mechanism appears to differ from that of the bacterial version of the enzyme, suggesting that it could be possible to design antibiotics that selectively block the bacterial enzyme.

“People have been trying to figure out whether there is something different enough that you could inhibit bacterial enzymes and not the human version,” says Catherine Drennan, an MIT professor of chemistry and biology and a Howard Hughes Medical Institute Investigator. “By considering these key enzymes and figuring out what are the differences and similarities, we can see if there’s anything in the bacterial enzyme that could be targeted with small-molecule drugs.”

Drennan is one of the senior authors of the study, which appears in the Feb. 20 issue of the journal eLife. JoAnne Stubbe, the Novartis Professor of Chemistry Emerita at MIT, and Francisco Asturias, an associate professor of biochemistry at the University of Colorado School of Medicine, are also senior authors. The paper’s lead authors are MIT research scientist Edward Brignole and former Scripps Research Institute postdoc Kuang-Lei Tsai, who is now an assistant professor at the University of Texas Houston Medical Center.

An unusual enzyme

The RNR enzyme, which is found in all living cells, converts ribonucleotides (the building blocks of RNA) to deoxyribonucleotides (the building blocks of DNA). Cells must keep a sufficient stockpile of these building blocks, but when they accumulate too many, RNR is shut off by a deoxynucleotide molecule known as dATP. When more deoxynucleotides are needed, a related molecule called ATP binds to RNR and turns it back on.

An unusual feature of RNR is that it can catalyze the production of four different products: the nucleotide bases often abbreviated as A, G, C, and T. In 2016, Drennan discovered that the enzyme achieves this by changing its shape in response to regulatory molecules.

Most of the researchers’ previous work on RNR structure has focused on the version found in E. coli. For those studies, they used X-ray crystallography, a technique that can reveal the atomic and molecular structure of a protein after it has been crystallized.

In the new study, Drennan and her colleagues set out to examine the human version of RNR. This protein’s structure, which turned out to be very different from the bacterial version, proved elusive using X-ray crystallography, which doesn’t work well for proteins that don’t readily crystallize. Instead, the researchers turned to an advanced form of microscopy known as cryo-electron microscopy (cryo-EM).

Until recently, cryo-EM typically offered resolution of about 10 to 20 angstroms, which might reveal the overall shape of a protein but no detail about the position and shape of smaller structural units within it. However, in the past few years, technological advances have led to an explosion in the number of structures achieving resolutions of about 3 angstroms. That is high enough to trace individual protein chains within the larger molecule, as well as internal structures such as helices and even side chains of amino acids.

Scientists already knew that RNR consists of two protein subunits known as alpha and beta. Using cryo-EM, the MIT team found that the human version of the enzyme forms a ring made from six of the alpha subunits. When ATP, which activates RNR, is bound to the enzyme, the ring is unstable and can be easily opened up, allowing the beta subunit to make its way into the ring. This joining of alpha and beta allows the enzyme’s active site, located in the beta subunit, to perform the chemical reactions necessary to produce deoxynucleotides.

However, when the inhibitor dATP is present, the ring becomes much more rigid and does not allow the beta subunit to enter. This prevents the enzyme from catalyzing the production of deoxynucleotides.

Designing drugs

Several cancer drugs now in use or in development target the human version of RNR, interfering with cancer cells’ ability to reproduce by limiting their supply of DNA building blocks. The MIT team has found evidence that at least one of these drugs, clofarabine diphosphate, works by inducing the formation of rigid 6-unit alpha rings.

This 6-unit ring is not found in the bacterial form of RNR, which instead assembles into a distinct ring containing four alpha subunits and four beta subunits. This means it could be possible to design antibiotics that target the bacterial version but not the human version, Drennan says.

She now plans to investigate the structures of other protein molecules that are difficult to study with X-ray crystallography, including proteins with iron sulfur clusters, which are found in many metabolic pathways. The microscopy work in this study was performed at the Scripps Research Institute, but when MIT’s new MIT.nano building opens, it will house two cryo-EM microscopes that will be available to the MIT community as well as other potential users in industry and academia.

“The technological advances that have allowed cryo-EM to get to such high resolution are really exciting,” Drennan says. “It’s really starting to revolutionize the study of biology.”

The research was funded by the National Institutes of Health.

Combatting chemotherapy resistance

Graduate student Faye-Marie Vassel investigates a protein that helps cells tolerate DNA damage, sharing her expertise with budding scientists to further STEM education

Raleigh McElvery
December 8, 2017

Combatting chemotherapy resistance

Person with long, dark hair and lab coat stares into microscope.

Graduate student Faye-Marie Vassel investigates a protein that helps cells tolerate DNA damage, sharing her expertise with budding scientists to further STEM education

Raleigh McElvery

 

Faye-Marie Vassel has a protein. Well, as a living entity, technically she has many, but just one she affectionately refers to as her own. “My protein, REV7.” And it makes sense — if you were hard at work characterizing a single protein for all six years of your graduate career, you’d be pretty attached, too. Plus, the stakes are high. REV7, which aids in DNA damage repair, could ultimately provide insight into ways to combat chemotherapy resistance.

Although Vassel’s mother trained as an OB/GYN in Russia before moving to the U.S., serving as what Vassel describes as a “quiet” scientific role model, Vassel spent her early childhood emulating her father, a social worker, and engrossed in the social sciences. She intended to one day work in science policy — until high school when she joined an after-school program at the American Museum of Natural History in New York City, and discovered an additional interest.

Here, Vassel took a series of molecular biology classes and met her first female research mentor, a postdoctoral fellow at Rockefeller University, who encouraged her to participate in another, more advanced science program funded by the National Science Foundation.

“I initially had my doubts, but just having that support changed everything,” Vassel says. “That was my first time doing research of any kind, and I got a sense of the sheer diversity of potential research projects. That’s also when I heard there was something called biophysics.”

From that point on, Vassel was hooked. As an undergraduate at Stony Brook University, she initially declared a major in physics before switching to biochemistry. Later, when it came time to select a graduate school, she was split between MIT and the University of California, Berkeley. As she recalls, MIT’s graduate preview weekend made all the difference.

“I had the chance to stay with biology students and speak with professors,” she says. “The whole experience made the department seem personal, and demystified the graduate school process by making it more tangible.”

She proposed a joint position between two labs: Graham Walker’s lab, based in Building 68, and Michael Hemann’s lab situated in the Koch Institute for Integrative Cancer Research. Walker’s lab focuses on microbiology, DNA repair, and antibiotic resistance, while Hemann’s lab investigates chemotherapy resistance in hopes of improving cancer therapies. After stumbling upon one of their joint papers, Vassel decided she’d like to combine the two.

“It’s invaluable to have both perspectives,” she says. “Mike’s lab just celebrated its 10th anniversary, while Graham‘s just had its 35th. It’s been interesting seeing the different ways they approach their respective research questions, because they were trained in such different scientific eras.”

Although Vassel is currently the only student formally working in both labs, the collaboration between Walker and Hemann, aimed at combatting chemotherapy resistance, has been ongoing.

Frontline chemotherapies, including one anticancer agent called cisplatin, kill cancer cells by damaging their DNA and preventing them from synthesizing new genetic material. Just how sensitive cancer cells are to cisplatin — and therefore how effective the treatment is — depends on whether the cell can repair the damage and bypass DNA-damage induced cell death. In some cases, cells increase production of “translesion polymerases,” which are specialized DNA polymerases that can help cells tolerate certain kinds of DNA damage by synthesizing across from damaged DNA or DNA bound to a carcinogen.

Vassel’s protein, REV7, is a structural subunit of one key translesion polymerase, and its expression is deregulated in many different cancer cells. As Vassel suggests, if one aspect of these translesion polymerases — say, the REV7 subunit — could be altered to hinder repair, then perhaps cancer-ridden cells could regain drug sensitivity.

Thanks to recently-developed CRISPR-Cas9 gene editing techniques, Vassel has removed REV7 entirely from drug resistant lung cancer cells, and watched as cisplatin sensitivity was restored. She also conducted rescue experiments, adding REV7 back into cell lines lacking the protein to see whether those cells become resistant to the drug once again. Most recently, she has been working in murine models to see whether REV7 has similar effects in a living system.

If her hypothesis is correct, REV7 would be a powerful target for drug development. Treatments that inhibit REV7, she explains, could be used in tandem with frontline chemotherapies like cisplatin to prevent resistance.

Since her foray into biology at the American Museum of Natural History almost a decade ago, Vassel has maintained her passion for science outreach. During her time at MIT, she has served as a math tutor for middle schoolers in the Cambridge public school system. She also volunteered as a science and math mentor for high school students, as part of a dual athletic and academic program founded by MIT.

As Vassel wraps up her final year of graduate studies, she is torn between completing an academic postdoc and indulging her early interest in science education policy.

“Growing up in New York City, it was not lost on me that — despite the city’s wonderful diversity — people from historically underserved groups were still missing from many science-related positions,” Vassel says. “It got me thinking about the dire need for policymakers to improve curricula to make science more inclusive of all life experiences. There’s this idea that science is apolitical when it’s really not, and that mindset can have detrimental effects on equity and diversity in science.”

Photo credit: Raleigh McElvery
Tania A. Baker

Education

  • PhD, 1988, Stanford University
  • BS, 1983, Biochemistry, University of Wisconsin-Madison

Research Summary

Our goal is to understand the mechanisms and regulation behind AAA+ unfoldases and macromolecular machines from the “Clp/Hsp100 family” of protein unfolding enzymes.  We study these biological catalysts using biochemistry, structural biology, molecular biology, genetics, and single molecule biophysics.

Awards

  • Margaret MacVicar Faculty Fellow, 2008-2018
  • National Academy of Sciences, Member, 2007
  • American Academy of Arts and Sciences, Fellow, 2005
  • Howard Hughes Medical Institute, HHMI Investigator, 1994
Celebrating a decade of interdisciplinary microbiology

The Microbiology Graduate PhD Program spans 50 labs across 10 departments and divisions, offering a broad approach to microbial science and engineering.

Raleigh McElvery | Department of Biology
December 12, 2017

Ten years ago, MIT launched the Microbiology Graduate PhD Program. Today, it boasts 28 alumni and 33 current students, and offers a broad, interdisciplinary approach to microbial science and engineering. Between five and eight trainees enroll each year and can choose among more than 50 labs spanning 10 departments and divisions — from biology and biological engineering to chemical engineering and physics.

Many diverse disciplines are rooted in microbiology. Basic scientists use microorganisms as model systems to understand fundamental biological processes. Engineers leverage microorganisms to create new manufacturing processes and energy sources. Even ecologists, biomedical researchers, and earth scientists dedicate their careers to investigating the role of microbes in our ecosystems, on our bodies, and on our planet. In sum, the study of microbiology permeates so many research areas that no single department at MIT could house them all.

The idea for an interdisciplinary microbiology program first came to Alan Grossman, head of the Department of Biology, while he was recovering from a heart transplant back in 2006.

“There were people scattered all over MIT who were doing microbial science and engineering, but there was no mechanism to connect them or give students outside those departments easy access to the labs,” Grossman says. “I began by talking to a few faculty members in order to gauge general interest, before pitching it to a handful of department heads and forming a committee. Everyone was very excited about it, and it really grew from the ground up.”

The Committee on Graduate Programs approved his proposal in May 2007, and the first cohort of eight students began in the fall of 2008. Martin Polz, co-director since 2015 and professor of civil and environmental engineering, sat on Grossman’s initial committee.

“MIT’s program is unique from most other microbiology programs because it’s so interdisciplinary,” Polz says. “Many microbiology programs across the country are associated with medical schools and focused primarily on pathogenesis. The students who apply here really appreciate the breadth of our program, and it has become a fixture at MIT over the years.”

Kristala Prather, co-director since 2013 and professor of chemical engineering, said the program also provides an opportunity to bring life scientists and engineers together to tackle research questions.

“I find there is a difference in the way engineers and scientists approach research problems,” Prather says. “Each approach has rigor, but having both perspectives breeds a richer set of discussions than just hearing from one discipline alone.”

During the past 10 years, Prather has watched a thriving and diverse community unite, spurred by a common interest in the microbial world.

Nathaniel Chu, who matriculated in 2014, said the program allows him to sample different disciplines while still maintaining a close affiliation with his advisor’s home department, Biological Engineering. As part of Eric Alm’s lab, Chu studies the interaction between the gut microbiome and immune system, and how imbalances in that delicate relationship can trigger conditions such as Type 2 diabetes, obesity, and inflammatory bowel disease.

“The program provides flexibility to explore your research interests, and my advisor has given me a lot of space to conceive and manage my own projects,” Chu says. “I’ve been able to interact with a diverse set of individuals within the microbiology circle, including clinical partners, immunologists, geneticists, bioinformaticians, and computational biologists.”

Jacquin Niles, incoming co-director, was a junior faculty member in Department of Biological Engineering when Grossman first proposed the idea. He says the students — past and present — are the heart of the program.

“A lot has changed over the 10 years the program has been in existence, but the caliber of students has remained consistent,” Niles says. “If I had to emphasize any particular aspect of the program, the students would be numbers one, two, and three. Each generation has been exceptional, and they are all very much on top of their research game.”

Michael Laub, co-director from 2012 to 2015 and professor of biology, adds that the early students deserve much credit for the program’s success. “They took a chance on a brand-new initiative, and as a result we ended up attracting ambitious, risk-taking, and creative folks who really paved the way for current students,” he says.

Alumni pursue a variety of careers, ranging from academia to industry. Some join existing institutions or companies. Others start their own.

Mark Smith PhD ’14 was a member of the second graduating class. Like Chu, he was one of Alm’s advisees, studying networks of gene exchange within the human microbiome, and building statistical models to determine the role of environment in various gut-related diseases. Smith went on to co-found a nonprofit organization known as OpenBiome, harnessing the microbiome to cure recurrent Clostridium difficile infections. In 2016, he co-founded another company, Finch Therapeutics Group, focused on scaling and commercializing clinical treatments for diseases rooted in the microbiome. In 2017, he was named to the Forbes 30 Under 30 list for science.

“OpenBiome and Finch Therapeutics were really a translation of the initial work that was done through the microbiology program, and a step toward developing those tools to improve human health,” Smith says. “The program taught me the foundational work I’ve come to rely on in almost every aspect of my job today.”

Like Smith, Jacob Rubens PhD ’16 aims to apply his training at MIT to help develop new products. After working in Timothy Lu’s lab — straddling the realms of biological engineering and electrical engineering — Rubens joined Flagship Pioneering, a company that starts, funds, and runs breakthrough biotechnology startups in Cambridge, Massachusetts. Rubens was also named to the Forbes 30 Under 30 list for science in 2017.

During the six years that Rubens was at MIT, he watched the microbiology cohort grow from roughly 20 to a force permeating more labs across campus than he could count.

“It’s heartwarming to see people bringing a microbiological perspective into all these different spaces, and influencing cutting-edge research across the Institute,” he says. “As a microbiology student, you become an integrator and synthesizer of many different viewpoints, and a node to foster cross-talk between disciplines.”

As Niles prepares to assume the role of co-director in July 2018 and usher in the program’s second decade, he intends to maintain its integrity and structure.

“The program has matured into what it is today thanks to a lot of previous, careful thought,” he says. “The students have indicated that there is a lot of value in the structure that we’ve refined over the years, and so my goal is to continue that positive momentum.”