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Philip Hanawalt

Philip Hanawalt: “What we don’t know is what is most important in science”

Stanford professor who discovered the DNA's repair mechanism upholds creativity in research

From Salvador

eduardo cesarPhilip Hanawalteduardo cesar

When he agreed to this interview for Pesquisa FAPESP, geneticist Philip Hanawalt warned: “I won’t know the answers.” This was not a refusal to be interviewed – on the contrary. For the professor from California’s Stanford University, what we don’t know is the most important element in science – it was curiosity about the unknown that led him to the field of molecular biology and this is what still keeps him going at the age of 77. He came to Brazil in September 2008 at the invitation of the Brazilian Genetics Society to the organization’s annual congress, held in the city of Salvador, State of Bahia, where he presented the latest findings of his pioneering work – which have had this characteristic for half a century.

Hanawalt started working with DNA in 1953, the same year in which the spiral staircase-shaped structure of this molecule which makes up the genes was unveiled.  In the 1960’s he discovered how the mechanism that remedies errors in the duplication of the Escherichia coli bacteria’s genetic material works. The genetic material of any organism is able to self-duplicate, but is unable to do so without making errors – which is the source of much of the genetic variation that appears in the course of evolution and in many forms of cancer; these errors are automatically corrected by internal devices found inside the cells. In the course of his career, Hanawalt continued studying these error correction mechanisms, referred to as repair mechanisms.  Among other things, Hanawalt proved that this repair mechanism is not homogeneous in the genetic material of each organism. His presentation at the congress in Salvador attested to the fact that, throughout his career, he has kept up with technological developments and nowadays is able to detect how molecules act almost as if he were seeing them.

The invitation to participate in the scientific event coincided with an important celebration.  Thirty years ago, he married Graciela Spivak, an Argentine geneticist whom he had met in 1977, during a course at the University of São Paulo. “The love of my life”, he stated in the lecture during the congress in which Graciela, who works in his lab, also presented her work in progress. “It’s a special occasion to be able to come back to the place where we met and to have this opportunity to interact with the lively and enthusiastic group of students and colleagues in your lovely country”,  said Hanawalt, who has two children from his first marriage and two from his second one.

In his opinion, encouraging beginning researchers to think, be creative and find their own paths is more important than scientific developments.

In 2009 you are going to celebrate the 50th anniversary of the publication of your first paper.  What was the subject matter?
Yes, my first paper was published in 1959. I was studying for my biophysics doctorate degree under Richard Setlow, at Yale University, and I had to conduct an experiment to measure the DNA, RNA and proteins in cells exposed to ultraviolet light. The objective was to study what happens to these molecules after bacteria undergo ultraviolet light radiation and I conducted this test to find a more sensitive method to detect DNA and RNA synthesis. We knew that ultraviolet light killed the cells and caused mutations, but we did not know what happened to the DNA, although we already knew that ultraviolet radiation interrupted the DNA’s replication.

The technology and knowledge that is available today did not exist 50 years ago. I have the impression that it was necessary to have a lot of faith to believe that the results of the experiments would actually reveal something about genetic material.  Is my impression correct?
I wouldn’t use the word faith.  I would say that we used the technology available at the time to find the answers to what we were looking for. Nowadays, I worry about the post-graduate students who simply access a catalogue to buy kits to purify and sequence the DNA. So they end up learning almost nothing about the details of the method, which might lead them to make mistakes when interpreting the results. At the beginning of my course on DNA replication, I talk about very simple, classical experiments that provided very important answers. I think it’s rather worrying that students nowadays are so enchanted with technology. A student asked me one day, “Can I work in your lab? I want to clone something.” I asked him what biological issue he was trying to solve and he told me he didn’t know – he just wanted to clone a gene. I explained to him that there are millions of genes to be cloned, and he would need to have a reason to conduct his research.  Perhaps cloning might not have been the answer to what he had in mind.

In spite of the advances provided by sequencing techniques, Brazilian geneticist Fábio de Melo Sene commented in his opening speech of the congress that this technique was a stumbling block for studies on evolution.  Apparently, researchers no longer make a distinction between evolution patterns and processes.  Do you agree?
Yes, I think technology is important, but it is merely a tool.  It’s possible to get information quickly that could not be obtained before.  The micro-arrangements, through which we can place 4 thousand Escherichia coli bacteria genes on a blade and wash it with the RNA produced by the bacteria, are a marvelous modern approach. The genes bond whenever the RNA finds a complementary DNA fragment. This is how we know which RNA molecules the cell produced.  So we can conduct a number of experiments and observe how the phenomenon works.  We can ask questions, for example, we can ask what happens when we shine an ultraviolet light on them, when we warm them, etc. Some companies sell pre-fabricated blades.  The danger is that this makes the procedure easier and leads to more money being spent on experiments without bothering to think about their meaning. My message to students is that they should think about the biological process that answers their question, go back to the basic principles and seek simpler ways to find the answer to a question.  They shouldn’t skip stages and use techniques just because the techniques are fashionable. One of my students told me once that the bacteria from his experiment had died.  I asked him how he could be so sure, if there had not been enough time to cultivate them. He answered that he had smelled the test tube. I hadn’t thought about that! E. coli bacteria emanate an odor when they grow – the odor disappears when they stop growing.  He had found the answer in the simplest way, it was not necessary to do anything else.

When the DNA structure was described for the first time, suggesting a mechanism for its own replication; did people imagine that the process involved so many errors?
I took my first biology course at the end of my undergraduate course, in 1953, and I learned that the DNA is one of the chemical compounds found in chromosomes. That same year, Watson and Crick published their first article describing the molecule’s structure.  When I started my post-graduate studies in 1954, everybody was talking about the DNA and how it replicated. If two DNA strings are complementary “I’ll call one Watson and the other one Crick “, all you have to do is separate the strings to produce a new Watson and a new Crick. Nobody thought about repairs because nobody imagined that the DNA changed very much.  It was known that the DNA underwent mutations, but the general belief was that mutations were something rare. The fact is that life could not exist if DNA could not repair itself. In fact, the DNA is not that stable, it is constantly repairing itself. The first repair mechanism was probably related to ultraviolet light, because life evolved on a planet without the ozone layer, where the damage to genetic material had to be repaired.

Where did the idea that the DNA could be damaged and commit errors come from?
The first clue was the existence of mutants.  Attempts were being made to identify the agents that damaged the DNA. There was a lot of interest in finding out why some cells are sensitive to ultraviolet light and others are not. So researchers started preparing cultures of more sensitive mutants. It was necessary to discover what happened to the DNA which resulted in damage caused by light radiation.  In our research studies, we used uncultivated cells to find out what the replication of the DNA was like after exposure to ultraviolet radiation and we discovered that the replication was the source of tiny fragments.  At the end of my doctorate studies, I was able to show that inhibition of the DNA synthesis is in proportion to ultraviolet radiation, but the replication recovers itself.  The work of physicists also had a major impact on this field.  If something happens, cells are able to deal with the problem.  In physics, we learn how to reduce questions to the simplest possible model that we can test.  Max Delbrück suggested that biology studies be based on viruses, which have the simplest known genomes and foresee what each part of the genome does [he was awarded the Nobel Prize in 1969 for this research study].

So the physics perspective permeated the study of biology?
There were two categories of biophysicists.  One category was comprised of those who studied physics and moved to biology to create the field of molecular biology.  I studied physics in my undergraduate and master’s courses. My intention was to become a biophysicist, but I had to study more physics before I could become one. The other category of physicists was comprised of specialists in crystallography – they were interested in the structures of the proteins. Watson and Crick didn’t create the double helix model from nothing.  To begin with, their discovery depended on the work of a very discreet scientist, Rosalind Franklin, who was studying DNA crystals and discovered a diffraction pattern through X-rays. When Crick walked by the lab and saw the images, as he was a physicist and was familiar with X-ray diffraction, he said:  “This is a helix!”

Nobody had seen a helix there?
No. X-ray diffraction is very difficult to interpret. However, if a person knows how it is generated, the person can see a basic configuration and knows that this is a helix. Rosalind Franklin did not get the credit she deserved.  This normally happens in the scientific world, more often to women than to men. The work women do is just as important as the work the men do, but the bosses are usually men.  That was the situation until some years ago.

Going back to the repair topic, is there an estimate of the damage caused to our DNA every day?
Each cell goes through 10 thousand to 50 thousand changes a day. So the repair mechanism works non-stop. During a one-hour lecture, each person will probably have undergone approximately 1 trillion depurinations, that is, 1 trillion guanines (one of the molecules that comprise genetic material) leave the DNA in the space of one hour.

It´s frightening.
It’s interesting and frightening, but listen to this: if there is a loss of guanine in the DNA, then obviously there has to be repair.  We didn’t have the technology to make the observations at the time when people believed that the DNA was stable. This kind of damage occurs spontaneously because the DNA is unstable. One trillion seems like a huge amount.  You have 1014 cells in your body.  This means that 1 guanine is lost for every 100 cells.

Is a flaw in the repair mechanism the main cause of cancer?
I would say that the common characteristic to cancer is genome instability.  Mutagenesis is one of the causes of cancer. Estimates are that it is necessary to have at least 5 or 6 successive mutations to form a malignant tumor. But this is a random number – it could be 10 or 12. In addition, there are various ways in which cancer appears. There are thousands of genes which, when they mutate, can represent a tiny steps in the direction of cancer. But some represent a bigger step. The p53 gene is altered in one half of the human tumors. So this gene has to be watched, it is important for apoptosis to occur in severely damaged cells. When the gene mutates, it no longer goes into apoptosis, and therefore the gene can undergo other mutations and this can create a tumor. If you add major environmental substances to this situation – cigarettes are on top of this list: the risk of a non-smoker developing lung cancer is 1 in 10,000; whereas the risk of someone who smokes three packs of cigarettes a day is 1 in 100. This is a risk that everybody should be aware of.  Variations also exist in different regions around the world.  In some countries where people don´t have refrigerators, food products are stored in holes in the ground and get contaminated by a type of mildew that produces alpha toxin, the chemical component known as being responsible for most cases of liver cancer. The field of environmental carcinogenesis seeks to understand which agents we have to worry about.  This is an important field which is sometimes overestimated. In tests, very high doses of a chemical product are normally used to provoke cancer in rats.  As a result, it is stated that this same product provokes cancer in humans.  Not necessarily! A few years ago, studies proved that saccharine, the sweetener that we put into our coffee, caused bladder cancer in male rats.  The same did not hold true for female rats or mice – male and female – and no epidemiological studies had been conducted on  humans proving that saccharine caused cancer in humans.

And the rats had been given extremely high doses?
Yes, extremely high. Later on, researchers discovered that what happens actually is that crystals form in the bladder, which, in association with a protein that is found in the bladder, irritate the bladder walls. Considering the extremely high concentrations, cancer develops because of the constant irritation, which causes the cells to die, causes an excessive proliferation of cells, and this is what causes the cancer.  The more cells proliferate, the higher the probability that they will undergo mutations. When this was discovered, I was a member of a committee in California in charge of preparing a list of carcinogens and saccharine was on that list. Later on, when I heard about these results, I suggested that saccharine be taken off the list. “We don’t remove items from the list”, the coordinator of the group told me. It took three years to remove the item, because these issues blow up into legal battles before any products are taken off the list.  In the team, researchers discovered that ascorbic acid – Vitamin C – produces the same crystals in rats.  So should we stop drinking orange juice? We would get scurvy. The truth is that the dose creates the poison.

Can knowledge about DNA repair help to develop therapies for cancer and aging?
In principle, I would say the answer is yes.  But first we have to discover the causes and make distinctions between the causes that we can and cannot control.  These are the kinds of studies we are developing at the moment. In some kinds of cancer, stretches of the DNA break and are transferred from one chromosome to another.  This is not caused by chemical agents, but by the natural characteristics of our DNA. This is quite rare; otherwise, we would all have problems. We want to study how the intrinsic aspects of the DNA contribute to the development of cancer in comparison to external agents.  In relation to environmental agents, we need to identify the potentially dangerous substances and then establish the doses that we have to worry about. Thus, we will be able to reduce the exposure to some substances to a reasonable level or avoid any exposure whatsoever, starting with cigarettes…

… which have to be avoided?
Exactly. Coffee, for example, has thousands of substances.  As far as I know, only 30 of these substances have been tested in relation to the possibility that they cause cancer in rats; half of these substances have produced positive results – approximately 13 or 14 cause cancer in rats, when consumed in huge quantities.  So does this mean that we shouldn’t drink coffee anymore? If we drink 15 thousand of cups of coffee a day, some of the chemical elements may cause damage. On the other hand, just to illustrate the complexity, some chemical elements have an anti-cancer effect.  We don’t know. Coffee might have 5 thousand anti-cancer substances that revert the effects of other substances, even if all the 15 thousand cups of coffee are not consumed.

Are any lists prepared that specify anti-cancer substances?
Sometimes, yes.  Not systematically, but it would be interesting to do so.  In relation to aging, I don’t believe there is too much information.  We still don’t know whether aging is caused by wear-and-tear or if it’s scheduled in the biological clock. We can certainly accelerate aging by damaging the DNA; there is some interesting recent information on how oxidative damage causes aging. We also have to consider the fact that aging is a different process in each organ; no single factor is the cause of everything.  We know, for example, that the skin ages less if you stay out of the sun.  So each organ deteriorates and ages at its own pace.  The C. elegans worm apparently has a programmed aging, as do yeasts.  This seems to be something very complex.  Perhaps we have a global clock which establishes the maximum lifetime and over which we have no control.

In your lecture, you mentioned that damage is sometimes part of how DNA works. Could you explain how this works?
The immune system has to generate 1 billion different kinds of antibodies.  So in a biological system programmed to make a mistake every 10 billion molecules, how is it possible to foresee something programmed to make 1 billion errors?

Does diversity stem from these errors?
Mutagenesis is diversity. In addition, there is transcription-dependent mutagenesis. It is interesting to produce as many errors as possible in the RNA messenger of a gene that produces antibodies, in order to have various different copies based on a single stretch of DNA. Does this seem impossible? Well, first you have to mutate the DNA, but there has to be an internal way of doing this, as it is impossible to make the DNA smoke cigarettes.  The simplest way is the desamination of cytosine, which, when it loses amines, turns into uracil, which does not exist in the DNA – it exists only in the RNA. When this happens, the repair system removes the uracil, because it wants to repair the error, but it was designed to err when it repairs something.

Does it substitute the error with anything, or only with the original amino acid?
That’s right. In addition, to make things more efficient, there is a protein named AID, which increases the desamination by approximately half a million times. This provokes mutation. During replication, the DNA opens up to provide access to AID, and this happens during transcription, which is very quick. If the transcription is slow, then the AID can enter and machine-gun the DNA: bang-bang-bang, instead of bang…bang…bang. It’s like in the film The Godfather, where the gangsters used an abandoned tollbooth to block Sonny’s car and machine-gun it…

You were also a pioneer in the sense that you proved that the repair is not homogeneous in the entire genome.
Well, this stemmed from the studies conducted by a post-graduate student, Mimi Zolan, who came to my lab around 1978. We were interested in whether the repair of the DNA was homogeneous or not, if some regions were better repaired than others.  She analyzed the alpha-DNA, which is a sequence of 179 nucleotides repeated several times.  She discovered that this alpha-DNA was not being repaired as well as the rest of the DNA. This was the first example of the differentiated repair of the DNA. This was related to the structure of chromatin and had nothing to do with transcription. When Mimi left – I think this was in 1982 – she said, “Why don’t you look at the active genes?” Very often people have the misleading impression that professors are the ones who have the ideas and guide their students.  They certainly do have ideas, but many important ideas come from creative students.  One of my recent and brilliant post-graduate students, Justin Courcelle, had the “heretical” idea that some of the so-called recombinant genes are not present to act in recombination. He conducted some experiments and showed that this could be true in relation to the blocking of the replication of the DNA. This provoked anger among the researchers who were working on recombination, who said, “How dare he say this?” Justin published an article in the PNAS journal, which was an excellent paper, in my opinion. But somehow I ended up on an e-mail discussion list and people were saying that the article was terrible.  But the terrible thing to them was that Justin was defying conventional thoughts. Do you think scientists are open to new ideas? No more than other people are.

So you studied the active genes?
Actually, the opportunity to study something is also something that comes up by chance. On the other side of the biology building, across from my office, Robert Schimke is studying the expression of a gene from the ovary cells of a Chinese hamster; this gene is amplified fifty times. It’s like the alpha-DNA, there are multiple copies of the same thing.  If we’re looking for something in a specific gene, it´s easier to find this something in 50 copies of the given gene than in one copy. We measured the repair in this fragment, but it was necessary to have a combination of people and ideas to achieve a successful experiment which showed that the gene is repaired more efficiently than the rest of the genome, because it is transcribed. We submitted the article to Cell journal – I felt it was important to send the article to a major publication – the article was rejected. So I called the editor and said I wouldn’t have sent the article to Cell if I had not been convinced that the referred discovery was a major one – it was the first report on the selective repair of an expressed gene.  He listened to me and said “OK!” Not everybody can solve these issues by calling the editor of the journal, but I have built up some credibility, I suppose. This goes back to the fact that new ideas, no matter how exciting they are, are not always necessarily accepted. People don’t like things that go against their paradigms or that in some way remove the glory from these paradigms. In my opinion, it’s important to protect the interests of students with ideas, so that they are not bowled over by egos and people who have been in the field for longer and who can suppress what these creative students do, or incorporate their ideas without giving them their due credit.

Do these attitudes go against science?
Of course they do.  Another thing that seems totally idiotic to me is that some people feel threatened by their former students, as if they were competitors.  If the students you trained are not successful, then this tarnishes your reputation.  A lot of my enthusiasm about science stems from the fact that I see people trained in my laboratory who have become successful.  This is a way for a scientist to achieve immortality, as it is impossible to live for more than 50 years after the normal age limit.

 You also have some academic offspring here in Brazil, don’t you?
Rogério Meneghini (currently at the Latin American and Caribbean Center for Information on Health Sciences) was my only post-graduate student from Brazil.  I have systematically tried to have students from as many countries as possible – 34 countries so far.  Not only students – I’ve had foreigners who washed the windows of my lab, and secretaries. Anyway, Meneghini came to my lab in 1973, stayed for a year – perhaps a little longer – and did an excellent job. Then he came back to Brazil and started studying the repair of oxidative damage to the DNA and continued doing a good job in this field. Carlos Menck [currently at the University of São Paulo] was his student. Because of this collaboration, in 1977, Meneghini invited me to give lectures at a six-day course.  That’s when I met Graciela – my wife – who came from Buenos Aires in her boss’s place.

So now you’re here on a personal and a scientific mission…
Yes, and this was unplanned. Of course the Brazilian Congress of Genetics was not scheduled to celebrate our wedding anniversary!

Have you maintained contact with what has been going on in Brazil in your field?
Actually, Menck is a very special person in this field. I must say he is an example in terms of his talent to motivate students. His students choose interesting problems to work on, very often with uncommon organisms. In this way they are able to get important information which is of great help to knowledge.  Commonly, everybody works with the same bacteria, namely, the E. coli. Most of what is known about repair in bacteria has been the result of working with this species. But when analyzing other organisms, as he has done many times, one discovers that not all bacteria work in the same way as the E. coli does. There are other ways of doing the same thing; we can discover new things with new organisms.

Is this the best way to achieve these discoveries?
Yes, being creative; looking outside the box. Menck does this very well and he keeps updated in relation to modern technology.  I became a scientist because I was interested in knowing how things work and also because I wanted to be a teacher.  I focused on molecular biology because this field was fascinating and growing with leaps and bounds. When I was a post-graduate student in Denmark, my advisor organized an international meeting called Molecular Biology; 40 people attended this meeting.  Nowadays, if you organize a molecular biology congress, you will see 50 thousand participants.  I really enjoy traveling around the world to attend international scientific congresses.  Thanks to my work in the field of environmental mutagenesis, I go to countries where congresses are being held not because science is at a high level but because those places have local environmental problems.  So I travel to very interesting places.  National frontiers do not exist in science; we make friends all over the world, in countries with different cultures, and we establish links and common communication, even if the languages are different.  It would be great if the way science operates could be used as a model for the relationship among nations – but go say this to politicians and lawyers.