Ever heard of four-stranded DNA?
 

Ever heard of four-stranded DNA?

It was during the 1982 Symposium on Structures of DNA. DNA molecule with many conformations was the main theme. The first session on June 2 was led by Alex Rich and other structural biologists. While listening to the presentations sitting among the packed audience in the Bush Lecture Hall my mind was running busy to figure out how to make sense out of the stem-and-loop structure of native mitochondrial DNA (mtDNA). The stem was not any thicker than double stranded region in the EM pictures that I have taken of the pearl millet mtDNA. Stem-and -loop structure of DNA being reminiscent of a transposable element stuck on my head for the past two years waiting for a good explanation.

It is highly unlikely, but by all means, if the stem-and-loop DNA were the real representation of a transposable element it would be very nice, because then the use of the terminal inverted repeats could be explained. Cruciform structure was the hot topic then to explain the utility of inverted repeats. Proteins such as DNA recombinase and transposase were known to mediate DNA recombination. I began to think that when inverted repeats can encounter their counterpart that close in the stem region, then complementarities between the two duplexes might be physically recognized.

How close do two duplexes have to be to recognize each other's complementary sequences? My eyes were fixed to the space filling model of B-DNA being displayed on the podium. There was one more such model brought by another crystallographer. I could not wait until coffee break when I walked to the front and asked for a permission of Tom Steitz to lay my hands on the models. I wanted to see if the two models could be brought together close enough to slide into each other's major groove and become of the same thickness as a double stranded DNA. Well, to my dismay, the major groove of standard B-DNA was not wide enough to let the other duplex slide in.

On Wednesday evening, dining hall of the Blackford Hall was sparsely occupied since speakers of the Symposium were invited to dine out by tradition at houses of the members of Long Island Biology Society. We were engaged in an idle after dinner talk. Scientists on my table were mostly from European countries. I was waiting my turn to talk. I changed the topic by asking "Has anyone ever heard of four-stranded DNA?" "Four-stranded DNA? No, I haven't." "No, I haven't either." My burning question about the possible existence of four-stranded DNA that had been lingering on my mind all the day fell flat. Someone on the next dinner table, however, overheard my questioning and came to our table. "Did you talk about four-stranded DNA?" "Yes, I did." "Why, are you interested in it?" "Yes, I am." "Well, I have published one article about a unique DNA structure in Science not long ago." Robert Hopkins and I walk out to the back yard lawn to get engaged into more serious talk about four-stranded DNA enjoying the evening breeze from the Cold Spring Harbor Bay.

I explained that I am preoccupied by the notion of four-stranded DNA based upon my EM pictures of some unusual shapes of mtDNA. Hopkins introduced me to his own Science paper and to Stewart McGavin's two papers in Journal of Theoretical Biology (1971 and 1977). For the first time I learned that there are a few people who talked about four-stranded DNA. This reinforced me that I am on the right track of thinking, but then I was anxious to know how much is already known about it by others. I hurried down to the library as I said good night to Hopkins. Library was still open. I was able to track down those references on four-stranded DNA. I decided to check as well the famous Watson and Crick's 1953 NATURE paper on double helical structure of DNA to find out for the first time that their paper was accompanied by two more papers, one by Morris Wilkins group and the other by Rosalind Franklin. CSHL was generous for visitors in making copies free of charge. I was able to copy a rather full collection of the earlier day references on DNA structure, which I had no easy access before. I was all set to take a journey into the wonderland of four-stranded DNA.

http://plaza.snu.ac.kr/~mglab/en_frame.htm

Four-stranded DNA defies any experimental approach

For the second half of the Symposium on structures of DNA, I felt 'the loneliness in the crowd'. I was struggling inside to figure out what would be the best thing to do next. If I were ever to speak up about the 'Four-stranded DNA' and claim for the Nobel Prize, I would be better come up with descent experimental evidences. But it was hopeless. These unusual conformations of mtDNA was rather scarce and not of the uniform sizes and shapes. It would be impossible to isolate the 'stem-and-loop' molecule in quantity. As soon as I attempt to purify the 'four-stranded' stem portion it would disintegrate. If you ever to talk about DNA structure, or any biological structure in
general, you should use the language of X-ray crystallography or NMR. However, there is no way to obtain a homogeneous solution of the 'stem' DNA, not to mention even growing any crystal out of it. Do I even know the name and function of the mtDNA molecule or gene? No, there is no single soul in the whole world working on pearl millet mtDNA, and I just started looking into it. And it is not as easy as maize mtDNA either.

What if I talk to people working on the model organisms such as phage lambda, E. coli, yeast, fruit fly, and maize? They already have transposable elements to test it on. No, they would not listen to me who comes out of nowhere. If it were that simple and easy they could have finished the story in a couple of months even before I begin figuring out what experiments I should be doing. During coffee breaks those 'established' scientists from 'Big Labs' formed lines on the pay phones calling back the home laboratory. They seemed to be yelling and screaming. "So, did you get the result yet? Good, I will be talking in the next session." "You better hurry up and do the test right away. So and so talked about that and it is all hot here."

It was hopeless. Making a story of four-stranded DNA based merely on an EM picture of stem-and-loop shapes of DNA was only a day dream. It was not a science. I had better give it up. On the other hand, it was too neat an idea to simply dump into a garbage can. Transposable elements were hot topics then. Every one was proposing his own model about the molecular mechanism about how transposable elements jump around and insert onto a new target site. Movable genetic elements all had common features of terminal inverted repeats. Upon transposition to a new site they caused a duplication of 4-9 base pair target sequence in direct orientation. It was generally understood then that an enzyme called transposase would recognize the terminal repeats, and facilitates transposition of the 'jumping gene' into a target site. The idea that terminal inverted repeats could physically make direct contacts in the major groove and recognize each other's repeat as complementary sequences was totally out of sight. Instead, cruciform structure was one of the favorite configurations to explain the utility of inverted repeats.

Double helical structure of DNA has been fixed in everyone's mind as a fact. It had been proven and worked fine as evidenced by the explosive development of molecular biology and genetic engineering. Even with the EM pictures of tight stem-and-loop DNA on hand it took me two whole years before it slowly dawned on me that the stem region might contain a four-stranded DNA.

Space-filling model

On my way home to Kingston, where I was a faculty at University of Rhode Island, I visited Tom Steitz at Yale University in New Haven, Connecticut. I wanted to have a look at his space-filling models of DNA and protein. Space-filling models he showed to me were the CPK models, the same kind the canonical B-DNA had been built of and shown on the cover of a text book of Molecular Biology of the Gene. The model was built to the atomic scale. It was heavy and was supported by central axis and transparent ladders. To build a stem-and-loop DNA many sets of those models would be needed. To my dismay, price of it was way out of my reach.

As I was checking through the piles of the mail in my office I found one brochure that caught my eyes. It was about an introduction of a new space-filling model made of rather tiny plastic units that was distributed by the publisher Academy Press (AP). The plastic model had some unique features. This AP model had bases and ribose units separately and were connectable with oxygen and phosphate atoms. Caps for van der Waal's radius were removable to reveal the backbones of DNA molecule. Unlike CPK model the AP model allowed bond angles adjustable at 5 degree increments and stayed fixed through out global manipulation of the DNA strands. Being a plastic model it was small and light, and easy to handle. Most important, it was cheap. I asked for an opinion of a biophysics professor in the Department of Chemistry about the plastic model and my interest in four-stranded DNA. His response was down right simple and clear. "It is energetically impossible. Don't waste your time on it." On the next day I visited the associate dean for research. I briefly explained to him about what I am up to with the DNA space-filling model. I somehow persuaded him to make his historical contribution on research by buying me the AP model of DNA.

I never took any class about X-ray crystallography nor about atomic torsion angles. No wonder I did not dare talk to any knowledgeable scientist lest he would talk me out of this absurdity. The entire tool I had were the books "Structures of Nucleic Acids," "Double Helix," and the reprints about DNA structure that I made the copies of at the library of Cold Spring Harbor Laboratory. However feeble the possibility to be the truth, it was too good and too important not to take the chance. In my saltbox house on the 36 Heritage Drive, Kingston, Rhode Island my solo game of the DNA puzzle began. The DNA model I made was too big to take pictures of. I put the model on a white blanket on the bottom of the basement and I took the pictures on the top of the stair case.