DNA metabolism DNA replication DNA 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 – proofreading More 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 Repair 2.Base Excision Repair 3.Nucleotide Excision Repair 4.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