Albert Lasker Basic Medical Research Award, 2015





For discoveries concerning the DNA-damage response—a fundamental mechanism that protects the genomes of all living organisms.

By Evelyn Strauss

The 2015 Albert Lasker Basic Medical Research Award honors two scientists for their discoveries concerning the DNA-damage response, a mechanism that protects the genomes of all living organisms. Evelyn M. Witkin (Rutgers University) established its existence and basic features in bacteria, and Stephen J. Elledge (Brigham and Women’s Hospital) uncovered its molecular pathway in more complex organisms. The details of the two systems differ dramatically, yet they share an overarching principle. Both coordinate the activity of a large number of genes whose products shield creatures from potentially lethal harm.

Throughout their lives, cells withstand an onslaught of insults to their DNA. External agents such as chemicals and radiation bash it, and it also sustains abuse from within when normal physiological processes blunder. In humans, such events deliver tens of thousands of genetic wounds every day. The DNA-damage response detects not only DNA anomalies, but also other dangers, such as interruptions in the DNA-copying process. Living creatures then implement a multi-pronged strategy to ensure survival.

Bacteria, for instance, ramp up their DNA-repair capabilities, halt cell division to provide time to mend damage, and amplify their mutagenic facility. At first glance, the third activity might seem to conflict with the first two, but evolution has covered many bases—boosting the microbe’s ability to fix DNA and also increasing variation within the population, thus enhancing adaptability.

Like bacteria, mammalian cells construct DNA-repair equipment and arrest division when they detect genetic peril. In addition, when the extent of injury overwhelms DNA-restorative capacities, the DNA-damage response sparks cell suicide. The organism thus maintains quality control and defends itself against cancer, an illness that is characterized by rampant genetic misconduct, including unbridled duplication of cells that carry marred DNA. Flaws in the human version of the DNA-damage response system cause diverse illnesses, including cancer, neurodegenerative disorders, and immune deficiencies.

Radiating insights
When Evelyn Witkin began studying the basis of radiation resistance at Cold Spring Harbor Laboratory in 1944, researchers had barely established that DNA was the genetic material. Radiation in the form of X-rays and ultraviolet (UV) light was known to cause inherited genetic changes, but the molecular nature of such mutations remained obscure, as did the method by which cells guard against them.

In her first experiment with bacteria, Witkin inadvertently uncovered naturally occurring Escherichia coli variants that resist radiation’s ill effects. She intended to induce mutations, but she had no experience with the techniques and used a UV dose that was so high, it killed almost all the microbes. Of the 50,000 cells that she put on her petri dishes, four grew. They had somehow overcome the parent bacterium’s unusual radiation sensitivity.

To divide successfully, a bacterium must create a partition that separates the two daughter cells, and UV exposure temporarily impedes this process. The parental strain that Witkin used in her studies exaggerates the usually brief delay that is typical of most bacteria. In the parent strain, she found, tiny amounts of UV light demolished the microbe’s ability to create the partition, and the creature never recovered. Consequently, long spaghetti-like filaments developed and the cells eventually died. The UV-resistant strain, in contrast, resumed division after a short lag.

Alert to SOS
In the 1960s, Witkin began uniting these observations with others. First, she noticed parallels between two previously unrelated behaviors. In addition to evoking filamentous growth of bacteria, UV awakens bacterial viruses called phages, whose DNA has settled silently into the bacterial genome. Other investigators had just shown that UV irradiation initiates phage activation by destroying a protein that normally restrains its genes.

By that point, Witkin had discerned that UV treatment of her parent E. coli strain stimulates production of a substance that hinders cell separation. Perhaps, she speculated, manufacture of this substance normally is limited by an inhibitor (commonly called a repressor) that resembles the one that hampers phage gene activation. Maybe the same UV-induced molecular apparatus incapacitates both of the inhibitors, thus prompting filamentous growth and phage activation. This idea gained support from another group’s observation that cells with a single genetic defect spur both processes. A common pathway seemed to link the two phenomena.

In the meantime, Witkin had generated key insights into the mechanism by which UV light causes mutations. Radiation by itself does not generate inherited changes; subsequent cellular functions are required, she found. UV light triggers chemical reactions within DNA molecules that disrupt its structure and render the genetic code uninterpretable at those spots. Unless the original DNA letter is restored, she proposed, a promiscuous DNA-replication machine—an enzyme that is sloppier than the only one known at the time, which stops cold when it encounters DNA damage—inserts a random, and often incorrect, DNA building block. Witkin thus predicted accurately that DNA damage stirs production of an error-prone copying enzyme that fosters mutagenesis long before there was direct evidence for it.

Furthermore, UV mutagenesis in E. coli requires a protein called LexA. Perhaps, she suggested in 1967, LexA normally limits production of the error-prone enzyme. Two years later, she implicated a second protein, RecA, in generation of UV-induced mutations.

In the early 1970s, Miroslav Radman and colleagues (Free University of Brussels) found that another UV-induced process—Weigle mutagenesis—also requires LexA and RecA, and they pointed out that phage awakening shares these features. Radman subsequently suggested that these phenomena and possibly UV-induced bacterial mutagenesis depend on error-prone DNA replication, which he called SOS replication in reference to the universal distress signal. Witkin soon established experimentally that UV-induced bacterial mutagenesis belongs to this cluster of SOS activities.

Invigorated by this growing constellation of commonly controlled behaviors, she and Radman scoured the scientific literature for more examples of UV-inducible functions whose activities depend on RecA and LexA. We now know that the SOS response (see Figure) switches on dozens of genes whose products contribute to a broad array of activities, including DNA repair and mutagenesis, that promote survival under stressful circumstances.


From yeast to humans
By the late 1980s, scientists knew that eukaryotes, whose cells (unlike bacteria) contain nuclei, also respond to DNA damage. This system’s mechanism, however, was opaque. In 1987, Stephen Elledge and his postdoctoral advisor, Ronald Davis (Stanford University School of Medicine), accidentally discovered that quantities of the messenger RNA for a yeast subunit of an enzyme called ribonucleotide reductase soar when DNA is damaged and replication is blocked. That behavior made sense, as the enzyme helps construct DNA building blocks, raw materials that are needed for DNA repair and synthesis. This observation and others inspired Elledge to suggest that a signaling system detects deviant DNA structures and, in turn, adjusts the activity of multiple genes whose products contribute to DNA synthesis and repair. Conventional wisdom held that triggers for signaling pathways had to come from outside rather than inside cells, and his bold idea was not initially accepted.

With his own students and postdoctoral fellows (initially at the Baylor College of Medicine and subsequently at Harvard), Elledge devised numerous genetic tricks to investigate his idea. By hooking up the genetic control region for a ribonucleotide reductase subunit to a reporter that indicates when it is active, Elledge identified yeast strains that carry altered versions of machinery in the hypothetical pathway. Some provoke DNA-damage-inducible genes in the absence of DNA damage, whereas others fail to rouse these genes in its presence.

The first class of yeast strains included one with a defective version of an enzyme that makes DNA. Because faltering DNA replication in this strain mimics the effects of DNA damage, the observation established that stalls in DNA synthesis can prod damage-stimulated genes.

The second, “DNA damage uninducible,” class of genes, included one (DUN1) that encodes a classic signaling enzyme, a kinase. Kinases activate or inactivate proteins by adding chemical adornments called phosphates to them, and they often fire sequentially, turning one another on and off. Dun1 itself gains phosphates in response to DNA damage, Elledge showed. The protein thus emerged as a strong candidate for participation in the signaling pathway that he had predicted, and thus generated support for its existence. Intact Dun1 is required to incite DNA-damage-inducible genes, has the enzymatic potential to transmit messages to downstream members of the pathway, and is activated in response to DNA damage.

Additional genetic strategies allowed Elledge to place more participants into the yeast pathway. He showed that DNA damage and replication stalls converge on a single protein, another kinase called Sad1/Rad53, that governs Dun1 activity; he also identified two kinases, Mec1 and Tel1, that act upstream of Sad1/Rad53. These proteins provided a potential link to mammals, as they belong to a molecular family that includes a human protein, ATM. Defects in ATM underlie a fatal disease (ataxia telangiectasia) whose symptoms include a high incidence of cancer, and cells from individuals with the illness fail to arrest division appropriately in response to DNA damage.

Elledge and numerous other investigators, including Michael B. Kastan (then at St. Jude, Memphis), Antony M. Carr (University of Sussex), and William G. Dunphy (California Institute of Technology), subsequently worked out key features of the mammalian DNA-damage response pathway and showed that it closely resembles that of yeast. These organisms utilize a series of related kinases to drive activities that protect themselves from threats to genomic integrity (see Figure).

Simple signal, complex effects
Elledge’s illumination of the human pathway revealed in 2001 that ATR, a mammalian ATM-related kinase known to be essential for the DNA-damage response, requires another protein to do its job. The second protein, ATRIP, led him to the mechanism by which the pair senses DNA aberrations.

A molecule called replication protein A (RPA) plays a crucial role in this step, Elledge discovered in 2003. It sticks to single-stranded DNA, a structure that is generated by multiple types of DNA lesions. ATRIP grabs RPA-coated single-stranded DNA, bringing along the ATR to which it is bound. This event kicks off the ATR arm of the DNA-response pathway.

Although numerous participants in this system had emerged, the total collection had never been defined. To probe this issue, Elledge exploited the fact that ATM and ATR add phosphates at known amino acid pairs within their target proteins. In 2007, he identified more than 700 proteins that receive phosphates from ATM or ATR in response to DNA damage. These proteins perform a tremendous range of activities (see Figure) that are crucial for health, and especially for preventing malignancy.

Creatures as diverse as bacteria and humans react to DNA damage by instigating a multifaceted physiological response that enhances their ability to endure the assault and thrive in its aftermath. Witkin and Elledge laid the conceptual and experimental foundation for our understanding of these intricately organized systems, which ensure genetic fidelity and safeguard organismal vitality.

Original Article : Lasker Foundation

Influential Interview with Amitabh Joshi : On evolutionary biology, and a passion for science

Amitabh Joshi
Amitabh Joshi (Photo: Amitabh Joshi)

Thoughts on experimental evolution, problem-solving and how to pursue science with passion.

By – Anjali Vaidya,IndiaBioscience


How did you become interested in evolutionary biology?

While I was doing my BSc Honours in Botany at Delhi University, I found genetics very interesting, because it brought back many of the things that I had liked about math and physics. There was a lot to understand, rather than just a lot to memorize. I remember being particularly impressed with how Jacob and Monod worked out the operon. I still think that’s one of the most beautiful things in genetics.

So after my BSc I applied for admission to MSc genetics at Delhi University. We had a course in population genetics the first year, taught by Professor C R Babu. He was quite simply the most amazing teacher I’ve ever had in my life. Many of the things he said in class I can still remember after almost thirty years.

Population genetics was just beautiful—it was cute, it was lovely. I really liked it. And so I decided to go for a PhD in evolution, and ended up working with Larry Mueller at Washington State University.

There was a certain amount of contingency in the choice of subject. I could as well have ended up a professor of Urdu literature or philosophy. I was reasonably clear that I wanted to be in academics. I couldn’t then and I can’t now imagine being in any other profession.

Can you describe some highlights of your current research?

The approach that we take in our lab is called experimental evolution. Instead of using an existing species to infer what might have happened in the past, you work with an organism that allows you to observe several hundred generations within a few years.

What we do is set up evolutionary problems for populations of fruit flies to surmount. For example, we took one set of populations and said that only those individuals that become adults at the fastest speed are allowed to breed for the next generation. After seventeen years and 600 generations, these populations are the fastest developing line of Drosophila melanogaster that anybody has ever seen.

When we asked what traits were sacrificed for the sake of developing fast, we found that these flies areworse than their ancestors at surviving to adulthood. We also found that faster developing flies preferentially burn lipids over carbohydrates as pupae. This gives them more energy per microgram, but it depletes their lipid reserves, which reduces fertility. In these populations you have to first be among the fastest 20% to emerge, and if you burn up your fat reserves and lower your chance of survival to develop quickly, you will probably still have higher fitness.

Our recent work follows up on our observations that different competitive strategies can evolve under conditions of high larval density. Our evidence suggests that when a culture vial’s food column is very short, the build up of waste means that even though food is scarce, feeding faster will not help larvae survive. We are developing our results into a broader view of competitive ability that emphasizes the interaction between environmental context and larval density in the determination of density-dependent fitness.

We have also shown that density-dependent selection can lead to the evolution of more stable population dynamics. In collaboration with Dr. Sutirth Dey (IISER Pune) we are trying to develop integrated models of the evolution of both density-dependent fitness and population dynamics. These results represent the first major conceptual advances in density-dependent evolution and population dynamics since Larry Mueller’s seminal work in this field in the 1980s and 1990s.

What is the most satisfying research problem you’ve worked on?

When I was a grad student, I became interested in the so-called cost of sex problem in evolution. I found that there was a huge debate that had been going on for twenty years in the literature about what the cost of sexual reproduction really was. John Maynard Smith argued that it was the cost of producing males, while G C Williams argued that it was the cost of genome dilution.

I remembered from my BSc that dandelions and other asexual plants produce partially sterile pollen. But in all the models of the cost of sex, people assumed that plants that produce asexual eggs either do not produce pollen, or they produce pollen in equal amounts and with equal fertility as sexual individuals. So I built a model, which would look between these two extremes, treating the output and fertility of male gametes as separate variables. It very quickly turned out that the cost of sex has both components—cost of male function and the cost of genome dilution. If you model only these two extreme cases, you will either find that the cost of sex is due to the cost of male function or that it is due to the cost of genome dilution. Between extremes, there are both components to the cost of sex.

This work eventually grew into several papers with Professor Mike Moody in the Journal of Theoretical Biology. In terms of satisfaction, this is one of the most satisfying pieces of work that I have done. It solved an old problem in a very clean way, by showing that what you are seeing as a problem is the fact that you’re not looking at the whole picture—you are just looking at two extremes of the spectrum.

In some sense the work was all the more rewarding because there was no need for me to do it. It wasn’t part of my thesis work, or work that somebody had told me to do. It just grew organically out of something interesting that I studied in a course, and then led to eventually something very nice.

What advice would you give to people starting out in science?

If you do your work with a certain passion, your career will take care of itself, and you will enjoy yourself. When you work, the rest of the world should stop. And I don’t mean that you should work 365 days a year. You cannot plan that “Tomorrow morning from 9:30 to 10:30 am I will think of a novel hypothesis.” All my life, I have never been an organized, steady worker, but when I work, at that time I’m completely focused on what I’m doing—whether it’s writing a manuscript or analysing a particularly recalcitrant data set. At that point in some sense the rest of the world ceases to exist.

Nowadays, people view science too much as a career. I wish people would view science the way a classical musician views music. Of course singers earn money by singing, but they don’t think of it as a profession the way working in a bank is a profession. If your only aim is to publish in high impact journals, that can give you some measure of success within the parameters of the system. But I feel there are much more interesting ways of getting one’s kicks in science.

What are the most frequent misconceptions that you encounter regarding your work? Which questions do you dread?

The question I don’t like, but get quite often, is how is your work important to the upliftment of human society. Science is seen too much through utilitarian lenses. The purpose of science is to understand. Harnessing that understanding to practical use requires a very different mind- and skill-set.

The most frequent misconception nowadays is that every causal explanation in biology must coalesce to one or a few genes. This position is brainwashed into students by the time they come to grad school and does a great disservice to biology.

Can you talk a little about your own approach to mentoring?

Mentoring, to me, is the most important thing any of us ever do as academicians. Mentoring is the essence of our existence as a link in the continuing chain of human knowledge. It is not about giving a grad student a good problem to work on: it is about helping the student discover his or her own good problem. As Khalil Gibran said so beautifully:If he (i.e. a teacher) is indeed wise he does not bid you enter the house of wisdom, but rather leads you to the threshold of your own mind.

Ultimately, mentoring is about sharing. You share the logic of how you approach a problem, probing for the right fault line at which to attack. Too often, research supervisors just tell students what to do without really explaining why, and if the student asks why, it becomes an ego problem for the supervisor because it is interpreted as having their judgment questioned. People need to be encouraged to discover their own style of doing science. It is important to transmit to the mentee a whole world view, rather than just a set of scientific techniques.

Alongside evolutionary biology, you have found time to write Urdu poetry and study Indian history. How do your other pursuits feed into your research? How do you balance time?

I don’t think poetry feeds directly into science, or vice versa. Perhaps, at a more transcendent level, the spirit of poetry informs the spirit in which I do science. Science has its own beauty—there are models and formalisms that are astonishingly beautiful, as well as experiments that are beautiful in a way that makes you go vaah as if you had just read or heard a superb couplet.

I don’t balance time well. I cannot do things like say I will work on a manuscript before lunch and write a poem or read philosophy after lunch. I work episodically on all things, whether science or other interests. When I am in the frame of mind for something I become immersed in it, to the exclusion of other things.

What are your most and least favourite parts of your job?

Most favourite: teaching. Least favourite: listening in meetings to self-important people pontificate about what ails Indian science.

What is the best advice you have ever gotten?

From my mother: Jo kaam karo, theek se karo, ya phir mat karo. If you do something, do it well. Otherwise don’t do it at all.

Original Interview : IndiaBioscience