Elledge, Stephen

Stephen J. Elledge

Brigham and Women’s Hospital

Witkin, Evelyn

Evelyn M. Witkin

Rutgers University

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

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.

Illustration of DNA damage

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.

by Evelyn Strauss

Key publications of Stephen Elledge

Elledge, S.J. and Davis, R.W. (1989). Identification of the damage responsive element ofRNR2 and evidence that four distinct cellular factors bind it. Mol. Cell. Biol. 9, 5373-5386.

Zhou, Z. and Elledge, S.J. (1993). DUN1 encodes a protein kinase that controls the DNA damage response in yeast. Cell. 75, 1119-1127.

Sanchez, Y., Desany, B.A., Jones, W.J., Liu, Q., Wang, B., and Elledge, S.J. (1996). Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science. 271, 357-360.

Sanchez, Y., Wong, C., Thoma, R.S., Richman, R., Wu, Z., Piwnica-Worms, H., and Elledge, S.J. (1997). Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation via Cdc25. Science. 277, 1497-1501.

Matsuoka, S., Huang, M., and Elledge, S.J. (1998). Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science. 282, 1893-1897.

Zou, L. and Elledge, S.J. (2003). Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science. 300, 1542-1548.

Harper, J.W. and Elledge, S.J. (2007). The DNA damage response: ten years after. Mol. Cell. 28, 739-745.

Key publications of Evelyn Witkin

Witkin, E.M. (1947). Genetics of resistance to radiation in Escherichia coli. Genetics. 32, 221-248.

Witkin, E.M. (1967). The radiation sensitivity of Escherichia coli B: a hypothesis relating filament formation and prophage induction. Proc. Natl. Acad. Sci. USA. 57, 1275-1279.

Witkin, E.M. (1969). The mutability toward ultraviolet light of recombination-deficient strains of Escherichia coli. Mutat. Res. 8, 9-14.

Witkin, E.M. (1974). Thermal enhancement of ultraviolet mutability in a tif-1 uvrA derivative of Escherichia coli B/r: evidence that ultraviolet mutagenesis depends upon an inducible function. Proc. Natl. Acad. Sci. USA. 71, 1930-1934.

Witkin, E.M. (1976). Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol. Rev. 40, 869-907.

Witkin, E.M., (1991). RecA protein in the SOS response: milestones and mysteries. Biochimie. 73, 133-141.

Witkin, E.M. (2002). Chances and choices: Cold Spring Harbor 1944-1955. Annu. Rev. Microbiol. 56, 1-15.

Award presentation by Titia de Lange

The oldest written texts are 5000 years old and are only known today because their Sumerian authors used clay tablets and kiln-baked their texts into permanence. The Etruscans hammered their best ideas onto gold plates, which also survived thousands of years. The progression to papyrus and paper often made written words short-lived and now the transition to electronic text has reduced that fleeting time to a New York minute.

What we write on our iPads is doomed to disappear fast, and baking our tablets in a kiln will not bring permanence. Of course the same changes that shortened the half-life of our texts came with enormous improvements in multiplication and distribution. But we may want to consider some mechanism to enhance the durability of what we write, even if most of it does not seem worth preserving.

Nature has had to solve the same problem. Its text is written in DNA, an extremely fragile medium, more like tissue paper than a clay tablet. Yet, some of our genes have been used for more than a billion years to make essentially the same protein. How do living cells ensure their genetic instructions remain readable to future generations of cells and organisms?

Nature’s solution to this instability problem is to repair its DNA text. Because most cells have two copies of the text, a corrupted version can be restored using the intact copy as a template. For this system to work, cells need an editor to monitor the DNA for every kind of change, e.g., for paragraphs that are broken off, sentences that are interrupted, words that are changed. But how is this done?

The Lasker Basic award is given to two exceptional scientists who discovered the answer for the two main types of organisms in our biological universe. Evelyn Witkin found the solution used by the bacteria, like the Escherichia coli in our gut, and Steve Elledge worked out how this is done by the eukaryotes, cells like ours, which carry their DNA in a separate compartment, the nucleus.

In the 1940s, Evelyn Witkin was a left-wing radical studying at NYU, where she demonstrated against NYU’s policy of sending their basketball team to the apartheid South after editing out their black players. In line with her politics, she turned down NYU for her graduate studies and selected a Russian geneticist at Columbia University, Theodosius Dobzhansky, as her thesis advisor. Dobzhansky worked on fruit flies, but Witkin wanted to work with E. coli, which, as she learned from a paper by Salvador Luria and Max Delbruck, could be used for genetics. Dobzhansky suggested that she work with Delbruck at Cold Spring Harbor Laboratory. She stayed at CSHL for more than a decade and later worked at SUNY Downstate University and Rutgers University.

Delbruck gave Witkin a UV lamp and told her to make mutations in E. coli. Not a carefully considered thesis project, but enough for Witkin, who started her career in science with the same independence, courage, and grit that later helped her negotiate this patriarchal world.

Although UV kills E. coli, Witkin was able to isolate a few mutant strains that survived. These mutants allowed Witkin to dissect the mechanism by which E. coli monitors its DNA. This is an amazing detective story. In the 1950s and ’60s, the DNA damage field extended into many aspects of bacterial physiology, including the release of bacterial viruses, induction of an error-prone type of DNA replication, and changes in cell growth. To most scientists, these phenomena were not obviously connected. But Witkin, with phenomenal deductive insight, understood that there was a central principle lurking behind them and provided data to back up her view.

Like Hercule Poirot, who at the end of the detective story gets everybody together in one room to baffle them with his analysis of the murder case, Witkin organized a small conference where she presided over the final ‘reveal’ moment of her field. That meeting established the concept of the bacterial SOS response, a term originating from the creative biologist Miroslav Radman. The central player in the SOS response is a protein called RecA, which can only bind DNA that is not in its usual double helix form, a clear indication that the DNA text has been corrupted. When RecA binds to corrupted DNA, it helps to inactivate gene repressors, which releases their target genes to express their protein products. Thus a wide array of genes spring into action after RecA has destroyed their repressors, and, by appropriately repairing the damage, ensures the fidelity and stability of E. coli’s genetic text.

Steve Elledge started his scientific career studying the SOS response as a graduate student with Graham Walker at MIT. But Elledge became more interested in how eukaryotes sense damage in their DNA, a question he pursued as an independent HHMI investigator first at Baylor College of Medicine and later at Harvard. Elledge, a virtuoso geneticist, worked out a way to isolate mutant yeast strains that responded differently to DNA damage, thus identifying, as did with others, a set of DNA-damage response genes.

But what do these genes do? Most researchers expected something like the bacterial SOS system, but Elledge hypothesized that eukaryotes use a different system, very much like a burglary alarm, with a detector that senses the damage and sends signals to control posts that alert the various DNA-repair crews. To test his idea, he used biochemistry to figure out what the proteins encoded by the DNA-damage response genes do. His key discovery was that these genes encode protein kinases, proteins that add phosphate groups other proteins, which is a signaling mechanism by which cells can quickly change the function of whole sets of proteins. He found that a single alarm arising from a protein kinase is sent to another protein kinase, which then amplifies it into a dramatic change in the cell’s physiology so that the DNA is repaired before the cell continues in its division cycle. Elledge also showed how this eukaryotic system senses DNA damage, once again using the editing principle that DNA that is not in a double helix points to a corrupted text.

Many of these DNA-damage response genes are now notorious because inherited mutations of them, as for instance in the case of BRCA1, dramatically increase the likelihood of developing cancer, while others play a role in aging or neurological disorders.

The discoveries of Witkin and Elledge opened up new fields and established basic principles that are now standard textbook material. But better than being in textbooks, which are electronic and therefore passing bits, their discoveries form the stones in the foundation of our understanding of how DNA is stably inherited over generations. Perhaps the next time we rescue our electronic data with a backup file, we will note it was Nature’s principle of sensing and repairing a mistake that got us to this point.

Acceptance remarks

Interview with Evelyn Witkin and Stephen Elledge

Video Credit: Flora Lichtman