DNA metabolism DNA replication DNA repair DNA recombination

DNA metabolism

DNA replicationDNA repair

DNA recombination

Map of E.coli chromosome

Meselson-Stahl experiment – DNA replication is semi conservative

DNA replication

Nucleases (eg DNases) degrade DNA

Exonucleases – degrade nucleic acids from one side of the molecule 5’ → 3’ or 3’ → 5’.

Endonucleases – degrade at any site in a nucleic acid strand or molecule

DNA replication is very accurate – proofreadingMore than 90% of DNA polymerase activity in E.coli is carried out by DNA Polymerase I.

DNA polymerase I is not the primary enzyme of replication but has clean-up functions during replication, recombination and repair.

DNA polymerase II – DNA repair

DNA polymerase III – principal replication enzyme in E. coli.

DNA polymerases IV and V are involved in SOS response (repair of extensive DNA repair).

Nick translation

Nick (a broken phosphodiester bond, leaving a free 3’ and a free 5’ phosphate) occurs where DNA synthesis is to start.

Sequence in the E. coli replication origin ori C

Initiation of replication

DnaA – recognises origin sequences and open duplex at specific site

HU – Histone like protein, DNA bending proteins, stimulates initiation

DnaB – unwinds DNA

DnaC – required for DnaB binding at origin

DNA ligase

Termination of chromosome replication in E.Coli

Ter - 20 base pair sequence

Ter sequence bind to protein Tus – Tus-Ter complex arrest replication fork

Separation of chromosomes

DNA Repair

1. Mismatch Repair2. Base Excision Repair3. Nucleotide Excision Repair4. Direct Repair

1. Mismatch repair

2. Base Excision Repair

3. Nucleotide Excision repair

4. Direct repair

DNA recombination

Meiosis

Homologous genetic recombination – (also called general recombination) involves genetic exchange between any two DNA molecules that share an extended region of nearly identical sequence.

Site-specific recombination – differs from homologous recombination in that the exchanges occur only at a particular DNA sequence.

DNA transposition – is distinct from both classes in that it usually involves a short sequent of DNA with the remarkable capacity to move from one location in a chromosome to another.

Homologous Recombination and Holliday intermediates

Effects of site-specific recombination

Genetically Modification

Transposons