1. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. E.coli systems and recombination: Determinants of diversity: Overall aims ML Nine/ten lectures with Key topics. Homologous recombination and DNA repair Role of methylation and repair. Role of Plasmids; control of replication, transfer and stability. Illegitimate recombination: transposons and integrons Regulation of DNA transposition. You should: Have a basic grounding for further reading and other systems covered in the course (e.g pathogens). Be able to critically read key papers in the area. Critically assess the development of ideas to date.

2. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. DNA Repair mechanisms This section will review: – The Role of DNA damage and its repair in the generation of genetic diversity in bacteria – The mechanistic links of repair with recombination systems covered earlier You should be able to discuss the effect of environment on damage and repair. For example: – Chemical and Radiation effects – Phagocytic damage in relation to pathogen survival in the host – Errors in replication and their repair.

3. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. How can DNA become damaged? Mismatched bases – Polymerase error rate about 1 in 10 4 (see later lectures) – Deamination of C to U leading to mismatch Missing bases. Hydrolysis of purine-deoxyribose bond leading to AP-site. Structural damage. Dimer formation. Broken phosphodiester bonds. Chemicals/radiation REPAIR MECHANISMS NECESSARY FOR SURVIVAL.

4. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Types of DNA Damage Summarised G A C T ds DNA Break Mismatch Thymidine dimer AP site Covalent X-linking ss Break C-U deamination

5. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. General repair mechanisms needed EITHER Reverse damage (e.g. PHOTOREACTIVATION) OR excise DNA and patch repair the region Photoreactivation: Discovered in Actinomycetes in 1949 UV - DNA Damage - Cell Death UV - Bright visible light - survival ! 3 Steps: Photolyase (encoded by phrA and phrB genes in E. coli) recognises distortion at dimer. Light activates photolyase Dimer cleaved

6. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. General repair mechanisms needed: Excision repair. Discovered first as a general mechanism in 1964 T- Phage HOST CELL REACTIVATION T-Phage suspension DNA Damage Repair - WT Repair Plaques ! UV

7. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Excision repair........ 5’ 3’ 5’ 3’ 5’ OR 5’ 3’ 5’ UvrABC 5’ 3’ 5’ Pol1 5’ 3’ 5’ 3’ 5’ 3’ 5’

8. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Other repair routes. Excision repair involves up to 20 nucleotides uvrA,BC (D) mutants very senstive to UV light Mismatch repair A from of excision repair. Dam methylase involved see later re: methylation N-glycolylase excision repair Uracil either misincorporated OR C deaminated to U Uracil N-glycosylase action TO give AP site AP endonuclease cut Patch repairs above

9. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. How does UV light cause mutations? Discovery of error-prone repair. RecA required for high level of UV mutagenesis UV dose of 2 µJ/mm 2 leads to 120 dimers Long patch Error-prone repair Post dimer initiation Trans dimer synthesis The SOS Hypothesis Radman 1974 originally proposed an inducible repair system Requires RecA and a regulator system

10. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. SOS System in E.coli Repair normally at low level lexA gene identified as a regulator Recombine normally But NO increased UV mutagenesis (ie 30 dimers produces no extra mutants). Higher doses required LOW DOSE - Error-free repair HIGH DOSE - Error repair INDUCED LexA is an autoregulated repressor Represses level of activity of many genes Collectively called DNA Inducible (din) genes Includes uvrA,B,C,D and sfi etc... RecA protease activity; Cleaves LexA Also CI repressor inducing lysis

11. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. SOS System in E.coli PO lexA PO recA PO din PO din PO din PO din PO din PO din PO din PO din PO din PO din Low level expression DNA inducible repair genes PO lexA PO recA HIGH level expression

12. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Post Replication / Recombination Repair recA mutants VERY UV sensitive uvrA similarly uvrA recA mutants VERY VERY UV sensitive recA mutants recover slowly in the dark uvrA mutants do not UV dose Dimers/ Genotype Phenotype µJ/mm 2 genome WT WT 5.0 3200 uvrA No excision repair 0.8 50 recA No recombination 0.3 20 recA, uvrA Neither 0.02 1

13. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Post Replication / Recombination Repair Stephen C. Kowalczykowski (2000) Initiation of genetic recombination and recombination- dependent replication. TIBS 25 – April 2000 Double-stranded-break repair – see transposition of Tn10 later Recombination-dependent replication Replication-dependent recombination SEE ALSO Key Reference in NATURE; STRUCTURE OF RecBCD complex Nature 11th Nov 2004 vol 432, 187-193