DNA - An overview

Page
7

Since the genetic information is stored in DNA, the replication of DNA is central to all biology

Slide 43

Semiconservative Replication of DNA

When Watson and Crick proposed the double helical structure of DNA with its complementary base pairing, they immediately recognized that base pairing specificity could provide the basis for duplication.

If the two complementary strands of a double helix separated, (by breaking the H2 bond) each parental strand could direct the synthesis of a new complementary strand.

That is each parental strand could serve as a template for a new complementary strand.

Adenine for e.g., in the parent strand synthesis of Thymine in complementary strand.

This mechanism of DNA replication is called semiconservative replication

Slide 44

In considering possible mechanism of DNA replication, three different hypothetical modes are apparent.

1. Semiconservative

2. Conservative

3. Dispersive

Slide 45

Conservative: parental double helix remain intact (is totally conserved) and somehow directs the synthesis of a “progeny” double helix composed of two newly synthesized strand.

Dispersive: Here, parental strand and progeny strand become interspersed through some kind of a fragmentation, synthesis, and rejoining process.

Slide 46

The Meselson – Stahl Experiment

They proved that DNA replicates semiconservatively in 1958 by the common bacteium E.coli.

Meselson and Stahl grew E.coli cells for many generations in a medium in which the heavy isotope of nitrogen N15 had been substituted for the normal, light isotope, N14.

The purine and pyrimidines bases in DNA contain nitrogen.

Thus the DNA grown on N15 will have a greater density (Wt. per vol.) than cells grown in N14.

Since molecules of different densities can be separated by equilibrium density gradient centrifugation, they proved .

Slide 47

The density of most DNAs is about same as that of heavy salts such as CsCl.

For e.g., the density of 6M CsCl is about 1.7g/cm3

E.coli DNA containing N14 has density about 1.710 g/cm3

Where as E.coli DNA containing N15 has density about 1.724 g/cm3

When a heavy salt solution such as 6M CsCl centrifuged at very high speed (30,000-50,000 rpm) for 48-72 hrs, an equilibrium density gradient is formed.

Slide 48

Meselson and Stahl took cells that had been growing in medium containing N15 for several generation (thus contained “heavy” DNA).