1. DNA metabolismDNA replicationDNA repairDNA recombination

2. Map of E.coli chromosome

3. Meselson-Stahl experiment – DNA replication is semi conservative

4. 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

5. 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).

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

7. Sequence in the E. coli replication origin ori C

8. 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

9. DNA ligase

10. Termination of chromosome replication in E.Coli Ter - 20 base pair sequence Ter sequence bind to protein Tus – Tus-Ter complex arrest replication fork

11. Separation of chromosomes

12. DNA Repair • Mismatch Repair • Base Excision Repair • Nucleotide Excision Repair • Direct Repair

13. 1. Mismatch repair

16. 2. Base Excision Repair

17. 3. Nucleotide Excision repair

18. 4. Direct repair

21. 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.

22. Homologous Recombination and Holliday intermediates

23. Effects of site-specific recombination

24. Genetically Modification

25. Transposons