The Vishnu of DNA

How Sankar Mitra discovered a family of enzymes that, like the Hindu preserver god, repair and maintain the genomes

by Karen Hede

When Sankar Mitra was a boy growing up in Calcutta, he listened to his grandfather tell tales of the great Hindu gods: Brahma the creator, Vishnu the preserver, Shiva the destroyer. Meanwhile, he learned of a parallel realm of science, in which universal forces continually create, transform, and destroy, only to create again.

Long after his initial fascination with science developed, Mitra learned from the literature that the biological creator, DNA, is not an inert monolith, nor merely a passive blueprint, as it is so often perceived. For this creator must continually hold at bay the forces of disarray--an onslaught of chemical invaders with the power to turn the all-important genetic code into a tumbling tower of Babel. The only thing standing between order and chaos, between Brahma and Shiva, is an elaborate system that has evolved to repair and maintain the genome. It is here that Mitra, along with others, recognized the Vishnu of DNA--the preserver of the genome.

In 2002, Mitra, professor of human biological chemistry and genetics and a senior scientist at UTMB's Sealy Center for Molecular Science, and his collaborators published two seminal scientific papers describing their discovery of a family of enzymes that are emerging as key players in DNA repair. In the papers, published in the prestigious Proceedings of the National Academy of Sciences and in the Journal of Biological Chemistry, the scientists demonstrated that the newly discovered proteins are responsible for repairing DNA damaged by highly reactive oxygen species in a process called oxidation.

Oxygen's Dark Side

Most people know that oxygen is essential, that it literally sustains our lives. However, oxygen's dark side is becoming more and more evident. "People worry about chemical damage and damage from UV light," says Mitra, "but the most dangerous chemical is oxygen. It is a paradox. We cannot live without oxygen, but at the same time we are constantly producing reactive oxygen species during respiration that damage our DNA. I always say, oxidative damage is the 'mother of all damage.'" In fact, Mitra has made a career out of studying how DNA defends itself from the destructive power of oxygen.

For some reason, good-oxygen-turned-bad--contained in derivatives known as reactive oxygen species, many of which are classified as "free radicals"--seems attracted to the four basic coding units of DNA: adenine, guanine, thymine, and cytosine. Typically abbreviated A, G, T, and C, these chemical bases form natural pair bonds--A with T and C with G--to make up the "rungs" of the classic DNA double helix. When they are damaged by free radicals, these bases no longer can bond correctly with their natural partner. Instead of pairing with C, a damaged G--called 8-oxo-guanine--now pairs with A. This case of mistaken identity jumbles the information encoded on the damaged DNA strand. Imagine this and many other, similar forms of damage happening over and over again, day in and day out, and you begin to get a sense of how important the systems that correct this DNA damage are.

"It has been estimated that as many as 10,000 oxidatively damaged bases are produced per cell per day," says Mitra. "If they are allowed to accumulate over a period of time, if they are not repaired, this damage leads to mutation." But most of us don't prematurely die of cancer or hardened arteries caused by such mutations, a fact which has led Mitra and others to an important insight: "We must have DNA base excision repair as a continuous part of normal living."

A Lesser God

In the pantheon of DNA repair, base excision repair (BER) might be considered one of the lesser gods. Studying BER, a kind of housekeeping DNA repair, traditionally hasn't had the scientific cachet of other types of DNA repair, such as nucleotide excision repair, which removes damage induced by ultraviolet light from sun exposure, and mismatch repair, which corrects mistakes created by the replication machinery itself.

"Base excision repair was always considered sort of the yawn, ho-hum pathway," says Stephen Lloyd, former professor and Mary Gibbs Jones Distinguished Chair in Environmental Toxicology. "If you knock out nucleotide excision repair then you have diseases like xeroderma pigmentosum, which makes folks hundreds to thousands of times more sensitive to sunlight, and if you have defects in mismatch repair, you are susceptible to various types of colon cancer."

Until the last year or so, however, no cancer or other disease had ever been associated with loss of a protein involved in BER. That fact alone may have turned some scientists away from the field. It is difficult to study a process when its effects are complicated and hard to trace. But starting with his co-discovery in 1980 of a peculiar enzyme called O6-methylguanine-DNA methyltransferase--also known as the "kamikaze" protein--which repairs damaged G bases but is itself destroyed in the process, Mitra has worked doggedly for more than twenty years to unravel the complexities of a system that he was certain must be essential for life.

"One of the fundamental truisms in the DNA repair business is that repair is universal, there is not a single organism that does not have a repair process," says Mitra. The so-called "lower organisms" in particular have a lot to teach us about basic biological processes.

It was discoveries in one of these lower organisms, the intensely studied E. coli, a bacterial colonizer of the human gut, that finally led to the discovery of a whole new family of base excision repair enzymes. By carefully searching the entire human genome, Mitra and his colleagues located two genes that appear to be evolutionarily related to E. coli's "nei" enzymes that repair oxidative damage in the bacterial genome. He named them the NEIL (nei-like) enzymes, an homage to their presumed evolutionary origin.

"Sankar has opened up the field of base excision repair," says collaborator Wah Kow, a microbiologist at Emory University and co-author with Mitra of the two papers that describe the new repair proteins. "One of the things he firmly believed is that just because loss of base excision repair enzymes does not immediately lead to cancer, doesn't mean they are not important. In these recent papers, he gives a very nice answer: there are a lot of back-up enzymes. This explains that in order to get a cancer, you would have to knock out several systems, and that's hard to do."

Since its discovery, one of these enzymes, Neil1, has been shown by Thomas Rosenquist and Arthur Grollman at the State University of New York at Stonybrook to play a role in the body's response to oxidative stress, which is thought to be at the root of tumor formation and the production of atherosclerotic plaque, as well as involved in the aging process.

"The jury is still out on the importance of the NEIL enzymes," says Lloyd. "But if these recent findings hold up, it would make the discovery important beyond the DNA repair aficionado. Now one would potentially see some direct implications on human health."

The Big Picture

In fact, for years Mitra and his colleagues have been studying BER and its implications for human health in minute, molecular detail.

In 2000, Mitra and collaborator John Tainer of the Scripps Research Institute, La Jolla, California, and their colleagues reported in the journal Nature that base excision repair is a highly structured, coordinated event in which the enzyme players perform a chemical hand-off from one step to the next, a kind of choreographed relay race so as not to leave a DNA strand dangling half-repaired. Although this analysis seems to make sense, historically most enzyme studies have been done in test tubes where conditions are highly controlled and variables limited.

"When I was a postdoc, we purified enzymes in order to study them," says Mitra. Following the paradigm of the day, the scientists sought to isolate and study enzymes in test tubes, away from unknown cellular factors that could complicate results. "Now we understand that we need to know how these enzymes interact inside the cell, how they are organized to carry out processes. This is the future of biology."

Mitra's interest in pushing the boundaries of his science has landed him in the middle of the largest program project ever funded by the National Cancer Institute. The $12 million, five-year grant's goal is nothing less than dissecting the entire structure, function, and cell biology of DNA repair. The program project, headed by John Tainer and Priscilla Cooper of Lawrence Berkeley National Laboratory in Berkeley, California, involves interaction among more than 14 scientists from a dozen different institutions including Harvard, MIT, Duke, and the University of California campuses in San Diego and Davis. While each project is a small component of the giant enterprise, the focus is on collaboration among various projects aimed at solving the structures of the complex DNA repair machines including that of BER, and their in vivo characterization.

"It's such a large problem that it can't be addressed in any one laboratory, by any one approach," says co-principal investigator Priscilla Cooper. "This program project brings together a very large collection of experts in DNA repair."

Cooper points out that it was Mitra's productive collaboration with herself and Tainer that helped convince the NCI to fund the project.

"He is always trying to put his science in a larger context that extends beyond the immediacy of his research," says Lloyd, whose own research on BER often intersects with Mitra's. "This interest in pursuing the larger questions of science becomes very infectious for students and young faculty. That's the reason he's a leader at the National Institute of Environmental Health Sciences center and has become involved in a number of large multi-center program project grants."

Says Cooper: "I've spent many long hours on the phone with him, tossing ideas back and forth about particular scientific problems. In these sessions with him the excitement is palpable. He has a bounce-per-ounce value that's way off the scale. He understands the big picture in a way a lot of people don't, and he is able to distill the essence of a lot of detailed work, to understand its global significance, and then to explain that to the larger scientific public."

Perhaps the most lasting tribute to his dogged pursuit of the complexities of BER is the groundswell of scientific competitors that has emerged. When Mitra began his study more than 30 years ago there were a handful of scientists working on BER. Now at UTMB alone there are other labs studying BER including Ella Englander, an associate professor of surgery, and Istvan Boldogh, an associate professor of microbiology. One of Mitra's erstwhile protégés, Tadahide Izumi, has become an independent investigator in the Sealy Center for Molecular Science after receiving a grant from National Cancer Institute to pursue his own studies in BER. "It [base exclusion repair] used to be more the sleepy side of DNA repair," says Lloyd. "But now it has become a very hot topic and is highly competitive." And Mitra deserves much of the credit for waking it up.

Karyn Hede writes about science and medicine from her home in Cedar Falls, Iowa.