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DNA Replication

27 Nov

DNA Replication occurs during the S (Synthesis) phase of cell cycle. The purpose of DNA replication is to create an identical copy of all the DNA in the cell so that, following cell division, both daughter cells will have complete copies of all the information required to build a cell and do all the things the cell does.

Data from several laboratories were elegantly integrated by the work of Watson and Crick to describe the structure of DNA as comprised of two anti-parallel strands bound together by polar (hydrogen) bonds between one purine and one pyrimidine.  Including:

1. Erwin Chargaff ‘s observations that

a) DNA was 50% purine (A and G) and 50% Pyrimidine (C and T) and

b) the proportion of A = the proportion of T; the proportion of C = the proportion of G .

2. Rosalind Franklin’s X ray crystallography data that indicated that DNA had a regular, repeating pattern and the molecule was of a specific width.

3. Oswald Avery’s group along with Hershey and Chase established that DNA was the genetic material (therefore making the structure of this molecule of high importance)

4. Knowledge of the distance between molecules engaged in hydrogen bonds.

5. Knowledge of the chemical properties of nucleotide molecules, comprised of hydrophilic deoxyribose sugars and phosphate groups and hydrophobic bases.

Altogether, this information provided enough background for the pair of researchers to arrive at the structure of DNA by engaging in model building.

How this all leads into the mechanism of DNA replication comes down to the following brief statement at the end of Watson and Crick’s Seminal Paper of the structure of DNA:

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”1

 

What did they mean by this?

“The novel feature of this structure is the manner in which the two chains are held together by the purine and pyrimidine bases…joined together in pairs, a single base from one chain being hydrogen-bonded to a single base from the other chain… [O]nly specific pairs can bond together. These pairs are: adenine…with thymine…, and guanine… with cytosine.”1

So, if the sequence of DNA bases on one strand dictates the sequence of the other, then each of the strands can be used as a template to make another. When this is done with each of the two strands, the result is two identical DNA molecules.

It’s one thing to say that it hasn’t escaped your notice that there is a mechanism for duplicating DNA inherent in its structure, but quite another to say that you know how it works.

This was the question that Matthew Meselson and Franklin Stahl were to solve in 1958.2 They imagined three possibilities:

  1. A Conservative method of replication – the original DNA splits open and new strands are made based on that information, then the original strands comes back together and the new strands zip together. We conserve both strands of the original copy.
  2. A Semi-Conservative method of replication – The original DNA splits open and new strands are synthesized to pair with each of the originals, the new DNA then exists with one original strand and one new one.
  3. A Non-Conservative / Dispersive method of replication – Frankly, I don’t know how this would work, but the result would be two new DNA molecules where bits of each strand of each molecule may be from the original or the new DNA.

How to distinguish between these methods?

Meselson and Stahl devised an experiment that in which they grew the bacteria, E. coli in broth containing DNA made of two different isotopes of Nitrogen. In one broth, let’s call it the ‘light’ broth, they had the light form of DNA with 14N, in the other, ‘heavy’ broth, they had the heavy form of DNA with 15N.

One really is heavier than the other. When they are centrifuged, they will come to rest at different ‘heights’ in the tube.

If the bacteria is grown in broth containing only the heavy DNA, and that DNA is harvested and spun down, you would see a tube like (a) containing a single band of the heavy DNA.

If than bacteria was moved into a new medium containing light DNA, and DNA was allowed to replicate once,

Assuming  semi-conservative or dispersive models of development – you would see (b) a single band of intermediate density – because all new DNA would be partly heavy and partly light.

Assuming the conservative model – you would see (c) two distinct bands – one heavy and one light.

So this immediately tests for or against the conservative model.

The actual result was a single intermediate band was found. This eliminates the conservative model of replication, but a second round of replication in the light broth is required to discriminate between those two models.

If the Semi-Conservative model is correct, then the intermediate band would remain, but a new light band would show up (d).

If the Dispersive model is correct, then the intermediate band would inch upwards (become lighter) as more light elements are mixed in randomly within the strands. (e)

What they found was exactly like that pictured in figure d. Further, if the bacteria were allowed to grow for more generations, the ‘light’ band of DNA would become larger as more light DNA is created, while the intermediate band will remain indefinitely.

 

 

Meselson and Stahl with Chase

 

 

References

  1. Watson. J. D. and Crick F.H.C.  “A Structure for Deoxyribose Nucleic Acid”  Nature 171, 737-738 (1953).
  2. Meselson, M. and Stahl, F.W. (1958). “The Replication of DNA in Escherichia coli”. PNAS 44: 671–82.
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Posted by on November 27, 2012 in Education

 

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