1. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Homologous Recombination Basic mechanisms Recombination in conjugation The Holliday Model Meselson Radding Model Recombination in intact replicons Enzyme complex involved Role of other genes Biochemistry of recombination

2. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Analysis of the rec system: How does the cell make it work? Some obvious points ! Mediated by enzymes within the cell. VERY efficient pairing for homologous DNA These in turn are encoded by genes in the cell. They have evolved with other cellular functions - are therefore interdependent. Their cellular environment must modulate their action. Environmental influence? All cells have the same substrate; DNA. Hence conservation of rec function likely in all organisms. You need to appreciate the complexity of the system to read further. The cell uses energy for this also!

3. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. A Complex of Enzymes involved Evidence for similar systems in other bacteria Some genes not always expressed in WT background Many associated with DNA repair - later Some under LexA/ SOS control - later Highly complex system operating ENVIRONMENTAL INFLUENCES ? LOGIC ? DNA - stable chemically Unstable within cell Acted upon by DNA encoded enzymes Generation of diversity by enzyme mediation Enzyme = chemical reaction = environmental influence

4. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Discovery of Recombination Genes Two basic approaches –Screen for recombination defficient mutants –Assess the recombination proficiency of other mutants (e.g. repair deficient) Recombination defficient mutants screened in Hfr crosses –Clarke et al (1965) PNAS 53, 451-459 –Howard-Flanders et al (1966) Screen for mutants unable to repair DNA damage –e.g from UV radiation –e.g from NTG mutagenesis

5. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Screening for rec mutants by Hfr crosses E.coli F- auxotroph Mutagenise NTG X-rays UV etc... x n E.coli Hfr MATE - + + -

6. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Classes of rec Mutant CLASS Map Position Recombination efficiency Sensitivity UV/X-rays rec A 58 min Below 10 -4 WT recombination VERY sensitive rec B 61 min 10 to 10 -3 WT recombination Much LESS sensitive rec C 61 min 10 to 10 -3 WT recombination Much LESS sensitive Highly PLIETROPIC. Sensitive to many DNA damaging agents such as NTG and alkylating chemicals Transduction defective No induction rec D also discovered 1984 & 1985rec BC also discovered as X-ray sensitive cells

7. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Recombination functions discovered rec A encodes a single protein rec BCD encode for an associated protein complex rec BCD mutants recombine at a low level: NOT so for rec A mutants LOW level pathway(s) present ? recA needed But recBCD not needed  recBC SUPRESSORS discovered (Clark et al 1980s) sbcA and sbcB

8. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Multiple Recombination Pathways Recombination or  WT(recA/BCD + ;recE/F + ;sbcA/B/C + ) recA    recBC (sbcA + ;recE + )   recBC (sbcBC + ;recF + )   recBC;sbcA (recE + ) recBC;sbcBC (recF + ) recBC;sbcBC;recF   recBC ;sbcA;recE   recBCD PATHWAY recE PATHWAYrecF PATHWAY

9. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Multiple Recombination Pathways……. What other rec functions are required ? recE and recF clearly. rec J N O Q R and others such as ruv discovered. Their full functions still not clear. Many other genes associated with the recombination process. See detailed handout. Clearly interdependent with other cellular systems. THIS IS AN IMPORTANT THEME

10. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Biochemical Properties of rec gene products. The rec A protein ---- Properties. Cloned and isolated in 1979 DNA dependent ATPase (ENERGY USED!) Protease activity (cleaves LexA repressor protein) Invitro activity ( starts with Flory 1985……….) Binds ssDNA role of ATP / ATPase activity Rapidly pairs ssDNA with homologous strands in a duplex Unwinds duplex DNA Promotes SLOWLY strand exchange and D - loop Minimum of about 40-50 bp homology needed

11. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Reactions promoted by RecA protein + ++ + +

12. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Reactions promoted by RecA protein…….. 3’ 5’ RecA protein forms a complex with DNA

13. ©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Reactions promoted by RecA protein Overview of recombination.…….. http://www.blackwellpublishing.com/trun/artwork/Animations/Recombination/recombination.html