DNA Replication

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Principle of complementarity

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The replication or duplication of DNA depends on one main idea, namely that the nitrogen bases of the nucleotides are complementary to each other on opposite halves of the molecule as shown here. Thus the base adenine(A) in DNA always pairs with thymine(T) and guanine(G) with cytosine(C). 

This pairing is due to the shapes of the nucleotide bases which allow hydrogen bonds to form between the complementary bases, holding the two halves of the DNA molecule together. 

This idea provided an immediate solution to how DNA replicated. All that should have to happen is the following sequence:

1. The parent DNA molecule unzips exposing the two halves of the DNA molecule

2. Each half of the parent DNA molecule serves as a template for the complementarity bases to be brought into the correct position to make a new complementary half for each of the original parent halves. This theoretical consideration suggested to Watson and Crick that DNA replication is what we now call semiconservative.

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Semi conservative DNA Replication

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Semi conservative DNA replication means that when the two new DNA molecules are formed during DNA replication, each resulting daughter molecule consists of one side from the original parent molecule and a new side synthesized from the parent side which served as a template.

 

The Meselson-Stahl Experiment:

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This experiment of Matthew Meselson and Frank Stahl demonstrated that DNA replication was semi-conservative. These scientists grew the bacteria E-coli in media containing nutrients with the isotope Nitrogen 15, 15N as opposed to the normal isotope of nitrogen, 14N. After a time some of the DNA containing the normal isotope is replaced with DNA containing ,15N as new DNA synthesis proceeds.

They cultured the bacteria for a sufficient amount of time that so that they could be sure that all the DNA contained just 15N. Then they took these bacteria and set up cultures using growth medium containing 14N and followed the bacteria through a number of replication cycles.

Meselson and Stahl then knew they could separate DNA's with differing amounts of 15N by using a process called cesium density gradient centrifugation which separates molecules by weight in a solution of cesium chloride. These scientists noted that one would expect a different distribution of DNA's by weight depending on whether the type of replication involved.

For example DNA replication could be conservative, in which case after one replication cycle half the DNA would contain only 14N and thus be lighter and the other half of the DNA would contain only 15N and thus be heavier. Thus there would be two DNA bands, one light and one heavy produced by the centrifugation.

DNA replication could have been dispersive in which case the daughter molecules after one generation would contain roughly equal mixtures of 15N and 14N mixed through each of the halves of the molecule, and there would only be a single DNA band of intermediate weight.

DNA replication could have also been(as does indeed turn out to be the case) semiconservative Again there would only be one DNA band of intermediate weight.

Meselson and Stahl realized that looking at only one replication cycle was not enough, given the equipment available to them in that the results could not distinguish between the dispersive and semiconservative mechanism. But if replication is allowed to continue for another cycle then the dispersive model predicts a single band of DNA, but the semiconservative model predicts a band of intermediate DNA and a band of light DNA containing only 14N.

The results matched the predictions of the semi conservative model but not the dispersive model. The difference in these models is shown in the figure.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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DNA polymerase:

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DNA replication in both prokaryotes and eukaryotes involves a complex of proteins including several enzymes called DNA polymerases. The DNA polymerases travel up the DNA molecule from an initiation site which is a region along the DNA that the enzyme complex can recognize.

 

 

 

 

 

 

 

 

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 pgd 7/29/02