Genetics - DNA structure

DNA is a polymer - series of repeated subunits

The subunits of DNA are called Nucleotides, and consist of three distinct parts

Phosphate group - phosphorous atom surrounded by four oxygen atoms - these form the connection between one nucleotide and the next - these have the advantage of being stable yet easily undone by enzymatic hydrolysis - necessary during replication and repair of the DNA.  The phosphate group always attaches to the 5' carbon of the sugar.

Pentose (5 carbon) sugar - ribose in RNA, deoxyribose in DNA - RNA has an oxygen atom at the 2' position, DNA does not.

Nitrogenous bases are the variable part of DNA and RNA

Purines - two kinds - Adenine and Guanine

Pyrimidines - Cytosine and Thymine in DNA, Cytosine and Uracil in RNA

A number of people worked on DNA structure from 1940 to 1953: Erwin Chargaff, Maurice Wilkins, Rosalind Franklin, Linus Pauling, Francis Crick and James Watson

Watson and Crick - ultimately proposed the correct structrue for DNA - double helix

Data- base composition analysis and X-Ray diffraction studies

Originally it was believed that DNA was made up of equal quantities of the four nitrogenous bases - this was known as the Tetranucleotide hypothesis.  Erwin Chargaff - analyzed the base composition of DNA in various species

Results:
1) amount of adenine is proportional to the amount of thymine in every species.  Amount of guanine is proportional to the amount of cytosine.

2) The sum of the purines (A + G) equals the sum of the pyrimidines (C + T)

3) C + G does not usually equal A + T

This refuted that tetranucleotide hypothesis

Maurice Wilkins and Rosalind Franklin - used X-Ray crystallography to analyze the structure of DNA - their work indicated that the molecule is a Helix.

Linus Pauling - proposed a triple helix

Watson and Crick used the X-Ray crystallography data and Chargaff's results, and developed a molecular model for the structure of DNA.

They found that a Double Helix fit the dimensions for the polymer derived from the X-Ray crystallography data.

1) two long polynucleotide chains coiled around a central axis

2) the chains are anti-parallel - their C-5 to C-3 orientatiions run in opposite directions

3) nitrogenous bases are perpendicular to the axis of the helix: they are stacked on top of one another, 3.4 angstroms apart, and are located on the inside of the structure

4) the nitrogenous bases of opposite chains are paired to one another as a result of the formation of hydrogen bonds - only A-T and G-C bonds occur.

5) Each complete turn of the helix is 34 angstroms long - 10 bases exist in each chain per turn

6) There is a major groove and a minor grove along the axis

The model required one purine and one pyrimidine per ring - these would be bound together with hydrogen bonds - stable hydrogen bonds would form between thymine and adenine (two bonds) and between guanine and cytosine (three bonds).  So GC pairs are more stable that AT pairs.

The relationship is called Complementarity.

Because of the higher stability of the G-C pairs, molecules that contain a higher percentage of G and C are more stable with respect to temperature.

One end of each DNA strand ends with a phosphate group on the 5' carbon of the last sugar, whereas the other ends with a 3' hydroxyl group.

Watson and Crick found that hydrogen bonding would occur if the polarity of the two strands ran in opposite directions - or Antiparallel.

Watson and Crick pointed out that the replication of the double helix could take place by unwinding the DNA, so that each strand would form a new double helix by acting as a template for the newly synthesized strand.

This mechanism of replication is called Semiconservative, because each new double helix has one old (template) strand and one newly replicated strand.

This is not the only possible mechanism of replication.  In Conservative Replication, the whole double helix would act as a temple for a new one, and one daughter molecule would consist of the original parental DNA and the other daughter molecule would consist of new DNA.

In Dispersive Replication, some parts of the daughter molecule would conserved, and some would not - so the daughter strands would  be patchworks of original and newly synthesized DNA.

Messelson and Stahl carried out an experiment in 1958 to test between these different hypotheses for the mechanism of DNA replication.

They grew bacteria (E. coli) in a medium containing15N, which is a heavy isotope of nitrogen (the normal form being 14N).  After growing the bacteria for

After growing in 15N for several generations, the DNA of the E. coli was denser.  The density of the strands was determined by using a technique called density gradient centrifugation.  In this technique a cesium chloride (CsCl) solution is spun in an ultracentrifuge at high speed for several hours.  This produces a density gradient, with the highest density of CsCl at the bottom of the tube.  An substance that is added will tend to concentrate at the point in the density gradient that matches its own density.  For DNA, the location of the DNA in the gradient can be detected with ultraviolet light at a wavelength of 260nm - which nucleic acids absorb strongly.