DNA - An overview

Page
9

Cairns interpretation of the autoradiographs was the semiconservative replication started at a site on the chromosome, which he called the, “origin” and proceeded unidirectionally around circular structure.

Subsequent evidence has shown his interpretation is incorrect on one point: replication actually proceeds bidirectionally , not unidirectionally.

Slide 57

Unique origin and Bidirectional replication

Cairn’s result provided no information as to whether the origin (the site at which replication is initiated) of replication is unique or occurs at random on the chromosome.

Moreover his results did not allow him to differentiate between uni - and bidirectional replication.

We now have direct evidence showing that replication in E.coli and several other organisms proceeds bidirectionally from a unique origin.

These features of DNA replication can be illustrated most simply and convincingly by experiments with some of the small bacterial virus.

Slide 58

Unique origin and Bidirectional replication

Bacteriophage lambda is like T2 a virus that grows in E.coli.

It has a small chromosome consisting of a single linear molecule of DNA only 17.5 µm long.

The phage λ chromosome has 12 nucleotides long at 5’end of each complementary strand.

These single stranded ends called, “cohesive” or “sticky” ends, are complementary to each other.

3’ 5’

G

GGGCGGCGACCTC

5’ 3’

Slide 59

Slide 60

The cohesive ends of a λ chromosome can thus base-pair to form a hydrogen bonded circular structure.

This conversion from the H2 bonded circular form to the covalently closed circular form is catalyzed by polynucleotide ligase, a very important enzyme that seals ss breaks in DNA double helices.

λ chromosome when replicates to circular form via θ - shaped intermediates.

Bidirectional replication was shows different at different segments like the region rich in AT and CG.

Schnos and Inman conducted an experiment on it using a technique called “denaturation mapping”.

Slide 61

When the DNA molecules are exposed to 1000 C or high pH (11.4), the hydrogen and hydrophobic bonds that hold the complementary strands are broken and two strands are separate.

This process is called denaturation.

Since, A-T region contains only 2 Hydrogen bonds it denature more easily than C-G

It denature to form “denaturation bubbles” which are detectable by electron microscopy, while C-G remain in the duplex state.

These denaturation bubbles uses as a physical markers whether the lambda chromosome is in its mature linear form or circular form or its θ -shaped intermediate .