Structural Biochemistry/Nucleic Acid/DNA/Chargaff's Experiments

Historical information
Determination of the DNA structure would not have been possible if it was not for the work of Erwin Chargaff, an Austro-Hungarian biochemist. Originally a scientist who did his first work in lipids and lipoproteins, after reading about an experiment of Oswald Avery which showed that DNA was material encoding the genetic information, he turned his work onto DNA. Tetranucleotide hypothesis was the mainstream theory on Chargaff’s time which was proposed by Phoebus Levene. The theory suggested “that DNA was made up of equal amounts of four bases – adenine, guanine, cytosine, and thymine – but that it was organized in a way that was too simple to enable it to carry genetic information.” The four bases are held together by hydrogen bonds and they are located inside the DNA helical structure. However, the sugar and phosphate backbone are on the outside of the DNA structure. The two strands are complementary to each other and thus one strand depends on the other. Despite the results of Avery’s experiments that DNA encodes life the scientific community was convinced DNA was relatively too simple to carry genetic information. Chargaff was not satisfied with the tetranucleotide postulation because of the minimal data that supported it.

Experiment
Erwin and his colleagues collected several DNA samples throughout their discovery. Using the fairly new technique of paper chromatography, Chargaff and his associates proceeded to separate DNA. The DNA that they collected was subjected to acid. The acid would then hydrolyze the phosphodiester bonds as it would cause a nucleophilic attack on the bond and result in the backbone breaking up. Once the phosphodiester bonds were broken then the individual nucleotides would then be separated and be free to analyze. Ultraviolet spectrophotometry was used to analyze the exact amounts of bases that were present in the DNA sample.

UV spectrophotometry showed that there was not an equal amount of purine bases (Adenine and Guanine) and pyrimidine bases (Cytosine and Thymine). Chargaff and his partners showed that the tetranucleotide hypothesis was in fact wrong in assuming that all four bases were in equal amounts. In other words, the concentration of GC equals to the concentration of AT. However, in RNA, Thymine is replaced with Uracil. What Chargaff noticed however was that although not all were in equal amounts certain bases were equal to each other. The base Guanine was equivalent to the amount of cytosine present; and the same held true for Adenine and Thymine. The ratio of A/T and C/G bases held true for all organisms and for both of the strands that were separated. The noticeable proportionality between one purine base to another pyrimidine base as well as it being true for both strands would be crucial in determining the helical structure of DNA although Chargaff was unable to see it.

The experiment gave two discoveries which is now summarized as Chargaff’s Rule:

1.	The number of Adenine bases is equal to the number of Thymine bases, and number of Cytosine bases is equal to Guanine bases. Ratio of A=T Ratio of C=G Ratio of A + T +C +G = 100%

2.	The proportion of A:T and C:G holds true for both strands.

For example: in human DNA, the four bases Adenine (A), Thymine (T), Cytosine (C), and Guanine (G) are present in these percentages: A= 30.9% and T= 29.4%; G=19.9% and C=19.8%. The A=T and G=C equalities, displays Chargaff's Rule, which actually remained unexplained until the discovery of the double helix by Watson and Crick.