1 MCB 502A - 2015. Lecture #2 DNA Structure. DNA denaturation / renaturation / annealing. The quest for structure of DNA What researchers new about the DNA structure before 1950: 1. In the 40s, it was realized that DNA could not be a tetranucleotide, because its molecular weight is huge and varies. DNA was imagined as long linear chains of four-nucleotide repeats. 2. Preliminary crystallographic characterization of DNA, done by Astbury in 1938, concluded that DNA material is very densely packed. To explain this DNA density, Astbury proposed that the DNA structure is a single chain with base and sugar pairs in the same plane and stacking. Besides introducing the biologically-critical concept of DNA base stacking, Astbury also correctly determined the distance between neighboring nucleotides at 3.4Å. 3. The mysterious Chargaff rule (A=T, G=C) the nature of which nobody, including Chargaff himself, understood. Around 1950, assault on the DNA structure began in Randall's laboratory at Cambridge University. Solving DNA structure is frequently attributed to Watson and Crick, but in fact it was a gradual and difficult process, 90% of which was accomplished by crystallographers. What we remember is the spectacular "discovery", but the drama and betrayal that surrounded it remain in the shade and darken the excitement. X-ray crystallography of simple molecules was all the rage at that time, but DNA was not only big, but like all filamentous molecules, was impossible to crystallize! Instead, a graduate student of Bernall, Sven Furberg, solved the crystal structure of a nucleoside cytidine, finding that sugar was perpendicular to the base. Therefore, Furberg proposed a model of DNA in 1952, based on a theoretical model of polycytosine monophosphate, with a single spiral strand, base stacking and sugars parallel to the axis of the spiral (in contrast to Astbury’s proposal, where sugars and bases were in the same plane). A postdoc of Randall, Maurice Wilkins, developed methods to make DNA into fibers, but the fibers, because they had to be thin (30 µM), were unstable, giving poor image quality, and Wilkins was only able to conclude that pure DNA is indeed a spiral. To get around the problem of DNA fiber instability, Wilkins tried X-raying cells, packed with DNA (like sperm cells), but image quality was still a problem (for a different reason now, — because DNA in cells is never “pure”), so Wilkins again just confirmed that “natural” DNA has the same spiral structure. Since the image quality precluded Wilkins from discerning more details about DNA structure, he eventually lost interest in the project, and Randall had to hire another postdoc for the task. This time, he was fortunate to stumble upon a new talent in the crystallography field, Rosalind Franklin. Franklin modified Wilkins’ method, by employing constant humidity chambers to stabilize DNA filaments by high humidity, and used it successfully to solve the DNA structure. Specifically, Franklin observed two forms of DNA: A-form at the 75% humidity, and B-form at the ≥90% humidity. A-form gave pictures with a lot of reflexes, while the more biologically-relevant B-form gave pictures with a few reflexes, appearing much less complex. Franklin, being a crystallographer, naturally concentrated on solving the structure of A-form (which, by the way, is similar to the current structure of DNA-RNA hybrids) and only touched on B-form. At this point, the influential theoretical attempt of Linus Pauling at solving the DNA structure should be mentioned. Pauling, of course, was the famous protein crystallographer who2 proposed the structure of alpha-spirals and beta-sheets, the two secondary structure elements that form the basis of any protein tertiary structure. The underlying philosophy of both secondary polypeptide structures of Pauling was the centrally-located backbone of the polypeptide repeats, with the variable parts of amino acids facing outward. That highly-intuitive logic seemed to fit perfectly biopolymers made of composite monomers, every such monomer having the constant part that polymerizes and the variable part that imparts unique properties on this monomer. The rationale behind these structures, “tuck in the backbone, stick out the variable determinants”, makes perfect biochemical sense for proteins. In fact, it seemed so naturally logical, that Pauling got epiphany and without any experimental evidence proposed a remarkable “biochemical” model of DNA. In this model, three sugar-phosphate backbones intertwined in a spiral braid, with the bases sticking out in all directions (the “branch-and-leaves” arrangement). It was a sequel to his famous polypeptide alpha-spiral and a beauty to behold. However, Franklin's painstaking calculations were revealing a totally different structure: for one, she calculated that both A- and B- forms were likely to be double-helices, rather than triple helices, in contrast to what was proposed by Pauling. Moreover, Franklin concluded that sugar-phosphate backbone must run on the outside in a wide spiral, rather than on the inside. She also positioned deoxyribose rings parallel to the main axis, like in Furberg’s model. The two groves of the helix were of unequal depth, but, importantly, the overall diameter of DNA was surprisingly uniform! For the A-form, Franklin also concluded that the two DNA strands run antiparallel. Since the A- and B- DNA forms were, essentially, the same DNA filaments, but at slightly different humidity, it was most likely that the two strands in B-DNA also ran anti-parallel. She was progressing steadily determining all the atomic coordinates of the reflex-rich A-form and was not in a hurry to publish her findings. Both to her credit and to her detriment, the insights into the DNA structure that Franklin was generating were openly discussed within the crystallographic circles at Cambridge. Specifically, Franklin shared her results freely with Wilkins, unaware of the fact that Wilkins was sharing Franklin’s results with whoever could sympathize with his own failure at the DNA structure... And here the story turns really interesting, almost bizarre. The main sympathizers turned out to be two theoreticians, Watson and Crick, who were also trying to understand the DNA structure, but who abhorred doing experimental work themselves and, therefore, were regarded as marginals. Crick, a physicist by training, was at least
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