THE THREE-DIMENSIONAL STRUCTURE OF DNA

It is clear that Deoxyribonucleic Acid (DNA), is the molecule in which the hereditary information is stored, investigators began to puzzle over how such a seemingly simple molecule could carry out such a complex function.

The significance of the regularities pointed out by Chargaff were not immediately obvious, but they became clear when a British chemist, Rosalind Franklin, acrried out an X-ray crystallographic analysis of DNA. In X-ray crystallography, a molecule is bombarded with a beam of X rays. When individual rays encounter atoms, their path is bent or diffracted, and the difraction pattern is recorded on photographic film.

Such patterns resemble the ripples cerated by tossing a rock into a smooth lake. When carefully analyzed, they can yield information about the three-dimensional structure of a molecule.

X-ray crystallography works best on substances that can be prepared as perfectly regular crystalline arrays. However, it was impossible to obtain trrue crystals of natural DNA at the time Franklin conducted her analysis, so she had to use DNA in the form of fibers. Franklin worked in the laboratory of British biochemist Murice Wilkins, who was able to prepare more uniformly oriented DNA fibers than anyone had previously.

Using these fibers, Franklin succeeded in obtaining crude diffraction information on natural DNA. The diffraction patterns she obtained suggested that the DNA molecule had the shape of a helix, or corkscrew, with a diameter of approximately two (2) nanometers, and a complete helical turn every 3.4 nanometers.

Learning informally of Franklin's results before they were published in 1953, James Watson and Francis Crick, two youthful investigators at Cambridge University, immediately worked out a likely structure for the DNA molecule, which we now know is substantially correct. They analyzed the problem deductively, first building models of the nucleotides, and then trying to assemble the nucleotides into a molecule that matched what was known about the structure of DNA.

Various possibilities were tried before they finally got the idea that the molecule might be a simple double helix, in which the bases of two strands pointed inward toward each other, thus forming base-pairs.

In their model, base-pairs always consist of purines, which are large, pointing toward pyrimidines, which are small, keeping the diameter of the molecule at a constant 2 nanometers. Due to the fact that hydrogen bonds can form between the bases in a base-pair, the double helix is stabilized as a duplex; DNA molecule composed of two antiparallel strands. The base-pairs are planar (flat) and stack hydrophobically, contributing to the overall configuration of the molecule.

The Watson, Crick model which can be seen at the top of this page explained why Chargaff had obtained the results he had: in a double helix, adenine will form two hydrogen bonds with thymine, however, it will not form hydrogen bonds properly with cytosine. Similarly, guanine will form three hydrogen bonds with cytosine, nonetheless, it will not form hydrogen bonds properly with thymine. Consequently, adenine and thymine will always occur in the same proportions in any DNA molecule, and so will guanine and cytosine.

HOW DNA REPLICATES

The Watson, Crick model instantly suggested that the basis for copying the genetic information is complementarity. Thus, one chain of the DNA molecule may have any conceivable base sequence, although this sequence completely determines that of its partner in the duplex.

Example; if the sequence of one chain is ATTGCAT, the sequence of its partner in the duplex must be TAACGTA. Thus, each chain in the duplex is a complementary mirror image of the other.

Replication Is Semiconserative

The complementarity of the DNA duplex provides a means of duplicating the molecule. In the event one were to open the molecule, one would need only to assemble the appropriate complementary nucleotides on the exposed single strands to form two daughter duplexes of the same sequence. This form of DNA replication is called semiconservative, because the original duplex is not conserved after one round of replication. Instead, each strand of the duplex becomes part of another duplex.

The existence of semiconserative replication was tested in 1958 by Matthew Meselson and Franklin Stahl of the Califorina Institute of Technology. The two scientist grew bacteria in a medium containing the heavy isotope of nitrogen, 15N, which was incorporated into the bases of the bacterial DNA. After several generations, the DNA of these bacteria were denser than that of bacteria grown in a medium containing the lighter isotope of nitrogen, 14N. The then transferred the bacteria from the 15N medium to the 14N medium and collected the DNA at various intervals.

The DNA collected immiediately after the transfer wer all dense. However, after the bacteria completed their first round of DNA replication in the 14N medium, the density of their DNA had decreased to a value intermediate between 14N-DNA and 15N-DNA.

After the second round of replication, two density classes of DNA were observed, one intermediate, and one equal to that of 14N-DNA. Meselson and Stahl interpreted their results as follows: after the first round of replication, each daughter DNA duplex was a hybrid possessing one of the heavy strands of the parent molecule and one light strand; when this hybrid duplex replicated, it contributed one heavy strand to form another hybrid duplex and light strand to form a light duplex.

Thus, that experiment clearly confirmed the prediction of the Watson-Crick model that DNA replicates in a semiconservative manner. Continue