The Story of DNA (Part 2)

We've discovered the secret of life.


.... Francis CrickWhere Jim and Francis Discover the Secret of Life

Following the disaster of their first attempt at a DNA structure, Francis Crick went back to studying proteins [The Story of DNA (Part 1)]. He and William Cochran worked out the theory of the X-ray diffraction pattern of helices. Crick became the leading expert on the interpretation of patterns due to helices and he was able to predict what kind of pattern a particular helical pattern would show. This study paid huge dividends later on. Crick also worked out the coiled coil arrangement of polypeptide chains.

Watson dabbled in a number of projects over the next year. Most importantly, he had Crick teach him diffraction theory and he applied it to the structure of tobacco mosaic virus showing that it was a helix. Watson too became extremely adept at recognizing helices from their X-ray diffraction pattern.

Franklin made one important discovery. She showed that there were two distinct forms of DNA and that the original Astbury pictures were composites of the two forms. She called them A and B and those are the same names that we give them today. The B form is the naturally occurring form and the DNA has to maintained at high humidity in order to persist in this form The A form is somewhat dehydrated, if the fibers dry out, the structure converts to the A form.

On April 10, 1952 Rosalind Franklin took a picture of the A form of DNA. This picture was complex but it had some significant new features. Franklin came to rely heavily on the images of dehydrated DNA (A-DNA). Over the spring and summer she convinced herself that DNA was not helical. In fact, on July 18th, 1952 she and Gosling announced “the death of the helix” by sending out small cards with black borders [Rosalind Franklin Anounces the Death of the Helix]..

Recall that Raymond Gosling was the former graduate student of Wilkins, now assigned to Franklin. Franklin was rapidly making herself a real pain in the you-know-what but everybody loved Raymond.

On May 2 and May 6, 1952, while immersed in the analysis of the A form of DNA, Franklin took two beautiful pictures of B-DNA (right). The photos screamed helix. A cursory glance by someone familiar with helical diffraction patterns showed that the bases were 0.34 nm apart; that there were ten nucleotides per turn; that each turn was 3.4 nm; that the phosphate groups were on the outside; that the diameter of the helix was 2 nm; and that there were most likely two polynucleotide strands. Rosalind Franklin did not recognize these features and she put the photos aside.

After announcing the death of the helix, Franklin set out to secure herself a new job at another institute. It is clear that she did not think that the structure of DNA was very important. She soon received an offer to move to Birkbeck College, London but delayed until the following spring. Meanwhile, she set herself the task of working out the structure directly from the X-ray diffraction patterns. She refused to engage in speculation or model building and preferred to try and let the data lead her directly to the correct structure. By January of 1953 she knew that she was not going to solve DNA and she prepared to abandon the problem and publish the data she had obtained.

With hindsight, it’s clear that Franklin needed a trusted collaborator in order to make progress on this difficult problem. While working in Wilkins group she found herself isolated because she and Wilkins did not get along. (Both were to blame.) Later on in her career she collaborated effectively with Aaron Klug and Francis Crick.

Franklin also proved that the unit cell of the DNA fibers was monoclinic, face centered. This was an absolutely crucial piece of information but one that Franklin failed to appreciate. As soon as Crick became aware of it, in January 1953, an important part of the structure became apparent (see below).

What were Watson and Crick up to in the summer of 1952 when Rosalind Franklin was announcing the death of the helix? Well, for one thing they were not ignoring DNA in spite of Bragg’s warning.

Crick had begun to consider the possibility that the bases might be on the inside of the helix. He asked his friend, John Griffith, to do some calculations to see whether the bases could interact with one another. Griffith replied that A and T are compatible and so are G and C. This has nothing to do with hydrogen bonds—that would come later—but it did confirm one idea in Crick’s mind. Both he and Watson were familiar with the idea of complementarity. Crick thought of it as a way of explaining how DNA was replicated since one part of DNA could give rise to its complement while the other could make the second part. Complementarity was often discussed among the phage group since Delbruck and Linus Pauling had published a paper on it just before the war.

They impressed me by their extreme ignorance … I never met two men who knew so little—and aspired to so much.

..... Erwin ChargaffErwin Chargaff visited Cambridge in May 1952. Chargaff met with Watson and Crick and explained his work on the base composition of DNA. The results were new to Crick but known to Watson. Crick immediately saw that A=T and G=C and that fitted in with his ideas about complementarity.

Chargaff was not impressed. He said later on that, "They impressed me by their extreme ignorance ... I never met two men who knew so little—and aspired to so much." Later on after the structure of DNA had been published Chargaff said, "That in our day such pygmies throw such giant shadows only shows how late in the day it has become."

After a round of conferences in the summer, life began to settle down again at the Cavendish labs in Cambridge. Watson and Crick were joined by two new members of the lab. Peter Pauling, the son of Linus Pauling, had become a graduate student and brought news from his father that Pauling senior was thinking about DNA. The other new member of the group was Jerry Donohue. He was a former graduate student of Pauling's who was joining Bragg's group as a post-doc.

On Wednesday, January 28th, 1953 a copy of the Pauling and Cory manuscript on the structure of DNA reached Watson and Crick. The structure was wrong. In fact, it was similar to the Watson and Crick model that Rosalind Franklin had destroyed fourteen months earlier. Watson and Crick were elated and they determined to try again in spite of the ban imposed by Bragg. (The ban was soon to be lifted.)

On Friday, January 30, 1953 Watson was in London and he stopped by to see Franklin in her lab (left). Watson showed her a copy of the Pauling and Cory manuscript and she too saw that it was wrong. Watson began lecturing Franklin about helices—remember that Franklin was, at this time, concentrating on the A form of DNA and had all but ruled out that it was a helix. However, she was beginning to have some doubts about her hasty announcement of the death of the helix [Rosalind Franklin Announces the Death of the Helix]. She resented Watson's lecture and advanced toward Watson with a view to dismissing him. Watson beat a hasty retreat. (Jim Watson is well over six feet tall and Rosalind Franklin is very much shorter.)

At that moment Wilkins came by and he and Watson walked off comforting one another in the knowledge that Franklin was impossible. Wilkins told Watson about the excellent pictures of B DNA that Franklin had taken eight months ago (May 1952). He showed Watson one of the pictures (see above). Watson left London with the knowledge that the B form of DNA was unmistakably helical, that the diameter was 2Å (2 nm), that there were 10 bases per turn, and that one turn was 34Å (3.4 nm). Some of this he got from the photo and some from measurements that Wilkins himself had made.

With this information, Watson started to build models. He began with the backbones inside but soon realized that it was impossible. Crick urged him to try to put the bases inside. Franklin had already concluded from her data that the phosphates were on the outside but it's not clear that Watson and Crick knew this.

Now comes a crucial bit of information. Rosalind Franklin had written a summary of her results for an institute report in December. Perutz gave Crick a copy. In that report Crick read for the first time that the crystalline form of DNA was based on a face-centered monoclinic unit cell. Why is this important?

It's important because such a unit cell has a two-fold axis of symmetry. That means that the molecule looks the same whether it is right way up or upside down. This has important implications for the two strands of DNA. To see this, think about two pencils side-by-side with the points down and the erasers on top. If you turn the two pencils upside down they look very different. Now the tips are pointing upward. However, if you line up the two pencils side-by-side with the tip of one pointing up and the tip of the other pointing down, when you flip the pair upside down they look the same. It means the two strands of DNA must be anti-parallel [The Chemical Structure of Double-Stranded DNA].

The space group of Franklin's DNA just happened to be the same space group as that of hemoglobin, the molecule that Crick was working on as the subject of his Ph.D. thesis. Crick recognized immediately what this meant.

Watson worked out another argument that convinced them that there had to be two chains in the unit cell and not three. It had to do with the density and water content and we won't go into it here. Suffice to say that in the last days of February they knew that the backbones were on the outside, that there were two chains, and that the chains ran in opposite directions.

On Friday, February 20th Watson presented some ideas about base pairs to his colleagues. He had come up with a scheme involving like pairs (A/A, G/G etc.). Jerry Donohue instantly recognized a problem. Watson was using the standard textbook structures of the bases, the imino and lactim tautomers. Donohue knew that the predominant forms in living cells were the other tautomers, the amino and lactam conformations [Tautomers of Adenine, Cytosine, Guanine, and Thymine]. This was the final important clue. Like pairing with like was not an option; besides, it didn't conform to Chargaff's rules.

The next week Watson made some cardboard cutouts of the bases and began to try and fit them together into the middle of the backbones running in opposite directions. Crick urged him to think about complementarity—recall that the previous summer Crick had convinced himself that complementarity was the key to DNA replication. He had forgotten about the A=T and G=C data from Chargaff.

On Saturday, February 28, Watson was playing with his cardboard cutouts when he discovered that you could fit A/T and G/C base pairs into the model. Crick immediately confirmed that this was an elegant solution. They then realized that it explained the Chargaff ratios.

It took them about a week to build a detailed model. Many experts were called to give their opinion and all pronounced it sound. Wilkins, Gosling, and Franklin came up to Cambridge to see the model and agreed that it must be right. Raymond Gosling is an admirer of Wilkins and in reviewing Wilkins' autobiography in Nature (Gosling, 2003) Gosling writes,

Wilkins eloquently describes his feelings at seeing the double-helix structure for the first time: "It seemed that non-living atoms and chemical bonds had come together to form life itself. I was rather stunned by it all." This sums up beautifully how Franklin and I felt. It was so elegant an explanation of all of the complex properties required of DNA, and contained so many elements familiar from our own work using X-ray diffraction. At the time I did not know that Wilkins was offered co-authorship by Watson and Crick, but refused. It would certainly have been appropriate, and seems to be something that he later came to regret.

The paper was written up and sent off to Nature on April 2. It was published on April 25, 1953 along with papers by Franklin and Wilkins.

Before publication, Linus Pauling visited Cambridge and confirmed that the Watson/Crick model was correct and his model was wrong. The first announcement of the discovery was made by Bragg at a conference in Brussels in early April.

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Franklin, R. and Gosling, R.G. (1953) Molecular Configuration in Sodium Thymonucleate. Nature 171:740-741. [PDF]

Gosling, Raymond (2003) Completing the helix trilogy. Nature 425:901.

Watson, J.D. and Crick, F.H.C. (1953) Molecular structure of nucleic acids. Nature 171::737-738. [PDF]

Wilkins, M.H.F., Stokes, A.R., and Wilson, H.R. (1953) Molecular Structure of Deoxypentose Nucleic Acids. Nature 171:738-740. [PDF]

Bibliography

Clayton, J. and Denis, C. eds. (2003) 50 Years of DNA. Nature/Pallgrave/Macmillan

Judson, H.F. (1996} The Eighth Day of Creation: Makers of the Revolution in Biology. expanded ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. USA

Maddox, B. (2002) Rosalind Franklin: The Dark Lady of DNA. Perennial/HarperCollins

Watson, J.D. and Berry, A. (2003) DNA: The Secret of Life. Alfred A. Knope, New York, USA

Watson, J.D. (1168) The Double Helix. Atheneum, New York USA

Nobel Laureates 1962

 
The other day we were talking about good American writers and the name "John Steinbeck" came up (for unknown reasons). This led to the obligatory question about whether anyone had actually read The Grapes of Wrath (some had) or The Log from the Sea of Cortez (nobody had). Anyway, it reminded me of a famous photograph I had once seen so I tried to find it on the internet. Here it is.

From left to right: Prof. Maurice Wilkins (Physiology & Medicine), Dr. Max Perutz (Chemistry), Francis Crick (Physiology & Medicine), John Steinbeck (Literature), James Watson (Physiology & Medicine), Dr. John Kendrew, (Chemistry). I hope Steinbeck was impressed.

Monday's Molecule #36

 
Today's molecule is complex. It has a strange-looking ring structure. The short common name of this molecule is well known but your task—should you choose to accept it— is to supply the correct IUPAC name. There's an indirect connection between this Monday's Molecule and Wednesday's Nobel Laureate(s). (The molecule also has a connection to Intelligent Design Creationism.)

The reward (free lunch) goes to the person who correctly identifies the molecule and predicts the Nobel Laureate(s). Previous free lunch winners are ineligible for one month from the time they first collected the prize. There's only one (Marc) ineligible candidates for this Wednesday's reward since many recent winners haven't collected their prize. The prize is a free lunch at the Faculty Club. The bonus is a free drink (alcoholic) with your lunch if you guess the connection to Intelligent Design Creationism.

Comments will be blocked for 24 hours. Comments are now open.

THEME:
Malaria
UPDATE The molecule is quinine, a drug used in the treatment of malaria. The Noble Laureates are Charles Laveran (1907) and Ronald Ross (1902)

Are you an atheist, agnostic, or a believer?

 
Blogger has a new feature so I thought I'd try it out. There's a poll in the left-hand margin. Click on the appropriate response.

Hillary Clinton Is a Marxist According to Mitt Romney

 
American politics is like a reality TV show. It can be very entertaining at times but it's sort of like watching a train wreck. Here's Mitt Romney telling us how Europe is in decline and America is great. Why? Because America promotes individual freedom and initiative while Europe went for socialism. According to Romney, Hillary Clinton is a Marxist who couldn't even get elected in France these days because Europe is voting for conservatives.

The Frequency of Alternative Splicing

One of these days I'm going to get around to blogging about alternative splicing. As most of you know, the databases are full of information about alternatively spliced gene products in mammalian genomes. There are many scientists who believe that most mammalian genes have two or more different products as a result of alternative splicing of the primary transcript.


I think it's nonsense. When I look at my favorite genes, the HSP70 gene family, the predicted protein products make no sense whatsoever. (The image shows predicted splice variants of the HSPA5 (BiP) gene from the SpliceInfo database.) The alternatively spliced variant often removes a piece of the hydrophobic core of the protein or other parts that are known to be essential. Since these proteins are the most highly conserved proteins in all of biology, it makes no sense at all to predict that mammals have all of a sudden evolved variants that are missing large hunks of highly conserved amino acid sequence. I think that most predicted splice variants are artifacts of the EST databases.

The annotators of the human genome have pretty much rejected all of the splice variants of HSP70 genes (e.g., Entrez Gene HSPA5) and many other genes whose structures are known. They have not rejected the multiple splice variants of other genes that are less well studied.

Anyway, like I said, this discussion will have to wait for another time. Meanwhile, you can read my friend Deanne Taylor's views on alternative splicing (and her disagreement with me) on her blog [Alternative Culture]. She will be here in Toronto next summer to debate the issue so everyone should plan on attending a mini-Howlerfest. It will be at the same time as the Darwin Exhibit at the ROM [Charles Darwin Is Coming to Toronto].

Here's an Example of Pro-War Thinking

 
Steve Huntley has a column in Today's Chicago Sun-Times [Careful, Iraq may be key to al-Qaida]. It's a really good example of the irrational thinking behind the pro-war crowd. Huntley begins with,

The Iraq war critics seized upon a new intelligence report that al-Qaida has been rejuvenated by the Iraq war as proof that the invasion of Iraq was a distraction from the war on terror. OK, that should be good for a few minutes of bashing President Bush, but it doesn't change the reality that al-Qaida is in Iraq and is our enemy.

No, the reality is that al-Qaida is now in Iraq and it wasn't before the invasion. The reality is that al-Qaida has been rejuvenated by the invasion and occupation of Iraq but we were told that this wasn't going to happen. We were told that the invasion and occupation of Iraq was part of the war on terror, but it wasn't really. It is now, because terrorism has been unleashed in Iraq since the breakdown of civil order. The breakdown of civil order was caused by the invasion and by the presence of foreign occupying troops.

Here's another thought: What would be the reaction of the quit-Iraq advocates should al-Qaida in Iraq's fingerprints be found in a terrorist attack in America?

This is not an idle question. After all, the National Intelligence Estimate released last week also said Osama bin Laden's organization will "probably seek to leverage the contacts and capabilities of al-Qaida in Iraq, its most visible and capable affiliate and the only one known to have expressed a desire to attack the Homeland." Furthermore, the 9/11 Commission has said another attack on America by Islamist terrorists is inevitable, and a new threat assessment a week ago from the National Counterterrorism Center suggested al-Qaida is working to renew attacks on America. Now we're told al-Qaida in Iraq could be the agent for it.

My reaction would be "I told you so." The invasion of Iraq has caused a huge increase in the number of people who hate America. Some of these people are going to be easy recruits to al-Qaida and some of them are, quite possibly, going to attack America.

Most of the damage has been done but we may be able to prevent further damage by getting the heck out of the Middle East and letting the people there solve their own problems their own way.

No doubt, even as the bodies were being recovered, the wounded treated and survivors consoled, the implacable Bush haters would blame his policies for an attack by al-Qaida in Iraq. But what would be the view of the majority of Americans who have been telling pollsters that it's time for America to withdraw the troops from Iraq?

I'm hoping that the majority of Americans would see the truth. It's the Bush policy that led to more people hating American and an increased probability that the war would be brought to America. I'm hoping that the impeachment of Cheny would be swift and that it would be followed by the impeachment of George Bush.

But then, I tend to be overly optimistic about these things.

[The image is from the US Dept. of Defense and is in the public domain. See Wikipedia: Army.mil-2007-02-13-104034.jpg]

Shalini Doesn't Like Appeasers

 
Shalini, over at Scientia Natura: Evolution and Rationality has written a longish criticism of appeasers [ Appeasers: The spineless pushovers]. Appeasers are also called accommodationists, they are atheists who do not want to criticize religious beliefs out of a mistaken impression that it's wrong and counter-productive to question another person's faith. The accommodationists believe that religion deserves some kind of protective status that they do not grant to people who believe in astrology, bigfoot, and UFO's.

You have to read Shalini's entire article to appreciate what she has to say but here's an important paragraph that I agree with. It gives you the flavor of her argument.

Contrary to what appeasers think, this is not about one issue or another. It is not about young earth creationism, ID, evolution, climate change, stem cell research, marijuana or the latest hot-button issue. These are merely battles in the course of the real war -- the war between rationalism and superstition. In this war, only one side will be the winner. There is no room for appeasers, and the superstitious, at least, will have none of this cowardly garbage. They may be ignorant, deluded, liars or plain kooks, but they are certainly not cowards, and that is more than I can say for the appeasers. Remember, no change has ever been achieved by shutting up and bowing down to oppressive institutions. If we fail to make our voices heard, superstition has already won.

Rosalind Franklin Announces the Death of the Helix

 
If you've read The Story of DNA (Part 1) you'll know that we've reached the point in November 1951 where Watson and Crick have failed in their first attempt. Rosalind Franklin was more than happy to point out how little they knew about the chemistry of DNA.

In Part 2 we'll learn how Watson and Crick redeemed themselves and how they we able to turn the tables on Rosalind Franklin. This notice, which was sent out to everyone in London and Cambridge who were interested in DNA, was signed by Raymond Gosling and Rosalind Franklin. It announces that DNA is NOT a helix. The date is July 18, 1952.

[The image is from a Francis Crick website.]

Ethidium Bromide Binds to DNA

 
Last Monday's Molecule was ethidium, better known by the name of its common salt, ethidium bromide [Monday's Molecule #35]. Ethidium is a large planer molecule that binds tightly to DNA. It is often used in biochemistry laboratories to visualize fragments of DNA that have been separated on gels. The ethidium molecule is fluorescent—when illuminated with ultraviolet light it shines in the visible range. Here's a picture (below right) of DNA fragments that are illuminated by ethidium binding. It's from an old paper of mine (Moran et al. 1979)—these days you usually can't publish simple experiments like this.

Ethidium binds by inserting itself bewteen the stacked bases in double-stranded DNA. Note that the ring structure of ethidium is hydrophobic and resembles the rings of the bases in DNA. Ethidium is capable of forming close van der Walls contacts with the base pairs and that's why it binds to the hydrophobic interior of the DNA molecule.

Molecules that bind in this manner are called intercalating agents because they intercalate into the compact array of stacked bases. In doing so, they distort the double helix and interfere with DNA replication, transcription, DNA repair, and recombination. This is why intercalating agents are often potent mutagens.

The cartoon below shows the distortion of the sugar-phosphate backbone when an intercalating agent bind and it also shows that the DNA is lengthened when intercalating agents bind. This changes the properties of DNA considerably. One of the tricks in separating closed circular molecules of DNA from linear fragments (such as genomic DNA) is to treat the DNA with ethidium bromide. The intercalating agent doesn't bind to closed circular molecules because they can't be lengthened enough to allow insertion of the chemical between the bases. The normal circular plasmid DNA can then be separated from linear DNA with bound ethidium because binding of ethidium changes the overall density of DNA.



The structure shown above (right) is from Reha et al. (2002). It shows a molecule of ethidium lying between two A/T base pairs.

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Moran,L., Mirault, M-E., Tissières, A., Lis, J., Schedl, P., Artavanis-Tsakonas, S., and Gehring, W. (1979) Physical Map of Two D. melanogaster DNA Segments Containing Sequences Coding for the 70,000 Dalton Heat Shock Protein. Cell 17:1-8.

Reha, D., Kabelác, M., Ryjácek, F., Sponer, J., Sponer, J.E., Elstner, M., Suhai, S., and Hobza, P. (2003) Intercalators. 1. Nature of stacking interactions between intercalators (ethidium, daunomycin, ellipticine, and 4',6-diaminide-2-phenylindole) and DNA base pairs. Ab initio quantum chemical, density functional theory, and empirical potential study. J. Am. Chem. Soc. 124:3366-76.

The Name of Buddy Holley's Airplane was "American Pie"

 
Friday's Urban Legend: FALSE

From americanpie.com.

Basic errors in American Pie interpretations have been carried forward and sometimes get reported as being fact. One of the most tedious theories of recent times is that the plane that crashed killing Buddy Holly, Ritchie Valens and the Big Bopper was called 'American Pie'. This is wholly untrue and Don McLean released a press statement in 1999 to confirm this:

"the growing urban legend that "American Pie" was the name of Buddy Holly’s plane the night it crashed, killing him, Ritchie Valens and the Big Boppper, is untrue. I created the term." - Don McLean, 1999

For those (one or two) of you who don't know what we're talking about, here's a video that interprets the song—one of the best pop songs of all time, especially for us old fogies who actually listened to Buddy Holly, Ritchie Valens and J. P. Richardson, Jr. (The Big Bopper).



[Hat Tip: Karmen at Chaotic Utopia]

200,000 Visits

 
According to Sitemeter I've had 200,000 visits to Sandwalk since I started last November. I have to admit that I'm fascinated by these numbers. It's exciting to see that some people are reading Sandwalk and it's fun to find out where they're coming from and which articles are the most popular.

When my six month trial period was up, I expressed some disappointment about the popularity of Sandwalk [My Six Months Are Up!]. Things look a little better now so I've decided to give it another six months and see how it works out. I don't know what I'll do when I run out of Nobel Laureates.

The Story of DNA (Part 1)

Where Rosalind Franklin Teaches Jim and Francis Something about Basic Chemistry

What we now know as DNA was first isolated in 1868 by Johann Friedrich Miescher, a student in the lab of Ernst Felix Hoppe-Seyler. A later student of Meischer’s named Richard Altmann called the material "nucleic acid."

Nucleic acids are composed of pentose sugars and four bases. Guanine was discovered in bird droppings in 1848 (guano means excrement of sea birds). Adenine was identified in beef pancreas in 1885. Thymine originally came from calf thymus, hence its name.

By the 1920’s it was clear that there were two kinds of nucleic acid, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), although they weren’t called by those names until much later. Chromosomes were known to contain DNA. It was thought that DNA was some sort of structural element that provided stiffening. The four bases were believed to be present in equimolar quantities and they repeated endlessly along the chain. This idea was referred to as the "Tetranucleotide Hypothesis." It was advanced by leading chemists and widely believed to be correct.

William (Bill) Astbury took the first X-ray diffraction pictures of DNA in the 1930’s. These first images, and Astbury’s interpretation of them, dominated thinking for fifteen years. Astbury noted that there were strong reflections at 0.34 nm and he interpreted this to mean that the phosphate groups were 0.34 nm apart. In other words, the repeating nucleotides were spaced at 0.34 nm intervals. Astbury concluded from his images that the bases were stacked on top of each other like a pile of coins. Both of these assumptions were correct. However, Astbury saw an important reflection at 2.7 nm suggesting the structure repeated every 2.7 nm. If DNA was helical then this would indicate that there were about eight bases in each turn of the helix and each turn was 2.7 nm. This turned out to be very misleading. Astbury also thought that the sugar moiety and the base were in the same plane and this is incorrect.

In 1944, Oswald Avery at the Rockefellar Institute in New York showed that bacteria could be transformed with pure DNA. This clearly indicated that DNA was the genetic material but the data was not widely accepted. The lack of acceptance was not due to Avery’s reputation since he was a recognized and highly respected scientist. It’s just that the concept of DNA as the genetic material didn’t fit with other data and couldn’t be reconciled with the supposed structure of DNA according to the Tetranucleotide Hypothesis. Nucleotides were known cofactors in metabolism and complex carbohydrates are usually structural as in cellulose and bacterial cell walls. Proteins, one the other hand, are special and unique.

Throughout the 1940’s everyone knew of Avery’s experiment but they set it aside as unexplainable. This is an excellent illustration of how science works. Usually it is a good idea to reserve judgment when a single experiment conflicts with the current paradigm.

Avery’s work stimulated Erwin Chargaff to take up the study of the chemistry of DNA. He carried out careful analyses of DNA from many sources and discovered that the base composition varied considerably. Some species had more guanosine and cytosine and less adenosine and thymidine while in others the relative compositions were quite different.

Chargaff also noted that the amount of adenosine was equal to the amount of thymidine and gaunosine equaled cytosine. It followed that the numbers of purines equaled the number of pyrimidines. For the most part, these molar ratios were not thought to be significant. What WAS important was the discovery that in DNA the four bases were not present in the same amounts. This destroyed the Tetranucleotide Hypothesis and paved the way to an important understanding: DNA could contain information now that the amounts of the bases could vary. It’s safe to say that only a small number of scientists appreciated this point. An even smaller number, not including Chargaff himself, appreciated the significance of A = T and G = C.

About this time James Dewey Watson was a graduate student at Indiana University in Bloomington, Indiana, He had gone there because of a famous geneticist, Hermann Muller, but Watson ended up in the lab of Salvador Luria working on bacteriophage. Luria, along with Max Delbruck, founded the ‘phage group—an elite group of scientists dedicated to discovering the secrets of life by working with the simplest organisms. They met every summer in Cold Spring Harbor and they visited each other often.

Watson’s Ph.D. thesis was unremarkable except for the fact that he completed it within four years and became Dr. Watson when he was only 21 years old. He set off to Europe on a post-doc. After a stint in Denmark with members of the phage group, he ended up in Cambridge.

After the war, an X-ray crystallography group was set up in Cambridge, England, to study the structure of proteins. The group was headed by Lawrence Bragg, a Nobel Laureate who developed the original theory of X-ray diffraction. The chief members of the group were Max Perutz, working on the structure of hemoglobin, and John Kendrew who worked with myoglobin. They later received Nobel Prizes for solving the first protein structures.

In the Fall of 1947 Francis Crick moved to Cambridge as an overage graduate student. He was 31 years old and had served in the Admiralty during the war. In the beginning he was associated with the biochemists and was in close touch with Fredrick Sanger. At the time Sanger was sequencing insulin. Although the results came in slowly over the next six years, Crick was aware of them instantly because he attended the monthly seminars. Sanger showed that every insulin molecule in beef pancreas had the same sequence of amino acids. This was the first direct evidence that proteins had a defined amino acid sequence and Crick realized right away that the sequence had to be encoded in the genes. Sanger got eh Nobel Prize for this work and later on received a second Nobel Prize for developing the technique of sequencing DNA.

1948: Linus Pauling was in Oxford as a visiting Professor on leave from the Californian Institute of Technology in Pasadena, USA. He discovers the α-helix and publishes the model with Richard Robert Cory on his return to California. This is the first clear indication of helices in macromolecules and soon after the model was published Max Perutz proves that hemoglobin contains α-helices. Pauling was the pre-eminent chemist at that time and his influence was enormous. He was in contact with the phage group but for the most part he was their major competitor. The fact that Pauling scooped the Cambridge group by coming up with a model for the &alpha:-helix will have an influence on later events..

1949: Crick moves to the Cavendish lab in Cambridge to study under Perutz. Crick had realized that it was important to learn X-ray diffraction in order to study structure. He had come to recognize the importance of information in the gene (whatever it was) and he hoped to discover how this information (sequence) gave rise to three dimensional structure. At this time Crick was interested in DNA but was not by any stretch convinced that it was the genetic material.

In the late 1940’s another structure group was established at King’s College in London, England. Maurice Wilkins joined the group and began to look at DNA fibers. In May of 1950, he had been given an excellent preparation of DNA containing large intact molecules (from Rudolph Signer in Bern, Switzerland). Wilkins’ first pictures were better than the fifteen-year-old images of Astbury. However, Wilkins’ interest in DNA was a sideline. The main focus of his work was proteins and he turned the DNA project over to a graduate student, Raymond Gosling.

Later on the group in London decided that they needed to hire someone more senior to work on the structure of DNA. They found Rosalind Franklin, a chemist who had been working on the structure of complex chemicals in Paris, France. She was anxious to return to England and when she arrived in January 1951 she immediately took over the DNA project, presumably under the direction of Maurice Wilkins. Wilkins gave Rosalind Franklin the excellent DNA samples that he had obtained from Signer the previous summer.

In retrospect, it is clear that Wilkins never meant to assign complete control of the project to Franklin. He was looking for a collaborator even though he assigned Gosling to Franklin as her graduate student. This conflict between Franklin and Wilkins became more intense over the next two years until they were barely speaking to one another.

Meanwhile, the evidence that DNA was the genetic material was mounting to those who were paying attention. The famous Hershey-Chase experiment was being completed and word was spreading among the insiders. In this experiment, Hershey had labeled bacteriophage DNA with radioactive phosphorus and the protein were labeled with radioactive sulphur. After the phage adsorbed to the bacteria, the culture was put in a Waring blender and the resulting agitation knocked off the empty phage particles. The bacteria could then be separated from the radioactive sulphur labeled proteins. When the bacteria were concentrated by centrifugation the DNA was found to be in the bacteria.

After a short time the bacteria lysed producing a new burst of phage. The experiment clearly indicated that the injected DNA carried the information to produce new phage particles. The Waring blender experiment was much more sloppy than Avery’s earlier experiment but it was confirmation and it got quite a lot of people thinking about DNA as the genetic material, especially those who were associated with the phage group and understood the significance of a phage head stuffed with DNA. Watson was one of those who realized how important the experiment was.

During the winter of 1950-51 Watson was in Naples doing some experiments when he attended a seminar by Maurice Wilkins who was visiting from London. Wilkins showed his X-ray diffraction images of DNA fibers. This impressed Watson who then decided that he had to learn about diffraction techniques in order to solve the structure of DNA. He managed to obtain a fellowship, with the help of his phage buddies, to study under Bragg in Cambridge.

October 1951: Jim Watson arrives in Cambridge and meets Francis Crick. Watson was 23 years old. Crick was a 35 year old graduate student. Watson convinces Crick that genes are made of DNA and together they resolve to discover the structure of DNA and the secret of life. The two became fast friends and spent hours talking about biology. They are moved to a separate office of their own in order not to bother anyone else.

It’s important to note how crucial this meeting was. Watson had convinced himself that DNA was the stuff of life and he needed to solve the structure. Watson had the biological background from hanging out with the phage group and the bacterial geneticists. Crick had taught himself about structure and X-ray crystallography and was certain that structures would provide clues to the secret of life. Watson and Crick were thinkers and talkers rather than experimentalists, especially Crick. At the time, Watson and Crick were among the few people in the world who really “knew” that genes were made of DNA. They may have been the only scientists who desperately wanted to solve the structure of DNA and achieve fame and glory.

Crick was a personal friend of Maurice Wilkins, the man who had taken pictures of DNA fifteen months earlier. Crick knew about the Wilkins’ pictures and he knew that Rosalind Franklin was making slow progress on solving the structure. Within a few weeks of Watson’s arrival they had constructed a model based on Astbury’s data; what Crick and Watson remembered of the Wilkins data; and what Watson had learned from Rosalind Franklin. Here’s how it came together.

Alexander Todd in Cambridge had just worked out the chemical structure of DNA. The backbone consists of alternating sugar/phosphate groups joined through the 3′ carbon of one sugar and the 5′ carbon of the adjacent sugar residue. Crick was also aware of the unpublished results of Sven Furberg, a graduate student in London. Furberg had solved the three-dimensional structure of cytidylate, one of the nucleotides in DNA. He learned that the base and the sugar were at right angles to each other. Recall that Astbury’s conclusion was that they were in the same plane and that view had dominated thinking for fifteen years. Furberg had proposed that DNA formed a single-stranded helix with the bases sticking out and stacked on top of one another.

Watson went to a seminar by Franklin in London on Wednesday, November 21, 1951. While there, he learned that DNA contained several chains, that the chains were probably joined by hydrogen bonds between the phosphate groups, and that the structure was a helix. Watson also thought that Franklin had said that each unit of DNA (i.e., one turn of the helix) contained eight water molecules. In fact, Franklin had said that each nucleotide was associated with eight water molecules.

In a few short days of feverish activity Watson and Crick had built a model of DNA. It had three chains and the phosphate groups were on the inside with the bases projecting outward. Watson and Crick invited Wilkins, Franklin, and Gosling up from London to see their triumph. Unfortunately for them, the first model was destroyed in a few minutes as Franklin demonstrated that it was impossible. She showed them that their structure did not have enough water and that there was no way to form the phosphate-phosphate interactions that they had modeled. Watson and Crick admitted defeat.

Shortly after this fiasco, Lawrence Bragg (their boss) ordered Watson and Crick to stay away from DNA. The problem belonged to Wilkins and Franklin in London.

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Bibliography

Clayton, J. and Denis, C. eds. (2003) 50 Years of DNA. Nature/Pallgrave/Macmillan

Judson, H.F. (1996} The Eighth Day of Creation: Makers of the Revolution in Biology. expanded ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. USA

Maddox, B. (2002) Rosalind Franklin: The Dark Lady of DNA. Perennial/HarperCollins

Watson, J.D. and Berry, A. (2003) DNA: The Secret of Life. Alfred A. Knope, New York, USA

Watson, J.D. (1168) The Double Helix. Atheneum, New York USA

The Three-Dimensional Structure of DNA

In order to understand the three-dimensional structure of DNA, it’s convenient to think of DNA as a ladder-like molecule with a very regular structure as shown below. The hydrogen bonding between base pairs is responsible for forming the regular structure with antiparallel strands.


The double-stranded helix forms as a result of interactions between adjacent base pairs. This interaction consists of van der Waals attractions and it causes the base pairs to come together so that they are in close contact. The result is a stack of base pairs, one on top of the other, with hardly any space between them. The usual term for these interactions are stacking interactions and they are the main force that holds the two strands together in a helical form.

The interior of double-stranded DNA is very hydrophobic which is just a fancy way of saying that water is excluded. Unlike proteins, the formation of double-stranded DNA is not an example of an entropy-driven hydrophobic effect, Instead it is enthalpic contributions in the form of stacking interactions that drive the reaction. However, the hydrophobic interior is essential because it stabilizes and protects the hydrogen bonds between the bases. These hydrogen bonds would not form if they were surrounded by water molecules since each of them could just as easily be replaced by hydrogen bonds with water.

The structure of the normal B form of DNA is shown on the right. B-DNA is a right-handed helix which means that if you think of it as a spiral staircase you will be turning to the right as you descend. Left-handed helices are found in some other rare forms of DNA. The width of B-DNA is 2.37 nm (nanometers) and it varies by only a small amount with base composition.

The distance between one base pair and the next is 0.33 nm, on average. This is called the rise as in the risers on a staircase. Some of the strongest reflections in the X-ray diffraction pattern of DNA are due to this repeat of 0.33 nm. The space between stacked bases is exaggerated in the cartoon (right).

The pitch of the helix is the distance to complete one turn of the helix. This value is usually given as 3.40 nm but it varies somewhat depending on base composition. On average, there are about 10.4 base pairs per turn of the helix in B-DNA and the angle of rotation between adjacent base pairs is about 34.6°.

Quoting from Horton et al. (2006)

The double helix has two grooves of unequal width because of the way the base pairs stack and the sugar–phosphate backbones twist. These grooves are called the major groove and the minor groove. Within each groove, functional groups on the edges of the base pairs are exposed to water. Each base pair has a distinctive pattern of chemical groups in the grooves. Because the base pairs are accessible in the grooves, molecules that interact with particular base pairs can identify them without disrupting the helix. This is particularly important for proteins that must bind to double-stranded DNA and “read” a specific sequence.

Two views of B-DNA are shown below. The ball-and-stick model (left) shows that the hydrogen bonds between base pairs are buried in the interior of the molecule where they are protected from competing interactions with water. The charged phosphate groups (purple and red atoms) are located on the outside surface. This arrangement is more evident in the space-filling model (right). The space-filling model also shows that functional groups of the base pairs are exposed in the grooves. These groups can be identified by the presence of blue nitrogen atoms and red oxygen atoms.


The length of double-stranded DNA molecules is often expressed in terms of base pairs (bp). For convenience, longer structures are measured in thousands of base pairs, or kilobase pairs, commonly abbreviated kb. Most bacterial genomes consist of a single DNA molecule of several thousand kb; for example, the Escherichia coli chromosome is 4600 kb. The largest DNA molecules in the chromosomes of mammals and flowering plants may be several hundred thousand kb long. The human genome contains 3 200 000 kb (3 x 10⁹ base pairs) of DNA.

©:Laurence A. Moran and Pearson/Prentice Hall 2007

The Chemical Structure of Double-Stranded DNA

 
Double-stranded DNA consists of two complementary polynucleotide chains where the bases on one strand form hydrogen-bonded associations with the bases on the other strand. There are only two pairs of bases that can form regular interactions where the edge of one base match the edge of another so that the two bases are joined by hydrogen bonds while lying in the same plane.

The A/T and G/C base pairs each have one purine (A or G) and one pyrimidine (T or C) which means that the size of each of the base pairs from one side to the other is almost the same. When two polynucleotide strands are laid side to side, as in the figure, the distance between the sugar residues on the two strands is the same for every base pair. What this means is that double-stranded DNA is a very regular structure in spite of the fact that the sequence of base pairs can be very different in different parts of the molecule.

The two strands are said to be complementary because all the bases in one strand are paired with the complementary bases on the other strand (A with T and G with C). This can only happen in a way that generates a regular structure if the two strands are antiparallel. If you look at the structure shown above you can determine the directionality of each strand by following the rules described in DNA Is a Polynucleotide. The left hand strand runs in the 5′→3′ direction from top to bottom while the right hand strand reads 3′→5′ from top to bottom.

The discovery of the structure of DNA by Watson & Crick was only made possible when they realized that the two strands of the helix had to be antiparallel.

There’s a convention for writing DNA sequences. They all have to be written in the same direction and that’s 5′→3′. Thus, the sequence of the bases on the left hand strand is AGTC and the sequence of the bases on the right hand strand is GACT. This may seem a bit confusing if you don’t understand the convention.

One of the classic questions on undergraduate exams is to give the sequence of one strand of double-stranded DNA (e.g., TAACTGGCGGA) and ask students to write down the sequence of the other strand. You’d be surprised at how many students haven’t paid attention when we discuss antiparallel strands in DNA and naming conventions.

©:Laurence A. Moran and Pearson/Prentice Hall 2007