©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Genetic Manipulation and Bacterial Genetics. M J Larkin

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Dr Mike Larkin - Communication MBC Room 108 –xt 2288 (Belfast Diverted to the DKB) DKB Questor Centre Room 316 – 3rd Floor Microbiology Laboratory – xt (9097)4390 or (9097)4388 lab’ or (9033) 5577 office Pigeon hole in Biology & Biochemistry office ALWAYS LEAVE MESSAGE and HOW TO CONTACT YOU! MBC Room 108 –xt 2288 (Belfast Diverted to the DKB) DKB Questor Centre Room 316 – 3rd Floor Microbiology Laboratory – xt (9097)4390 or (9097)4388 lab’ or (9033) 5577 office Pigeon hole in Biology & Biochemistry office ALWAYS LEAVE MESSAGE and HOW TO CONTACT YOU!

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Questor Microbiology and Environmental Genomics Lab’

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Course Theme. A detailed understanding of the molecular principles that are the basis for the generation of genetic diversity in bacteria. Basic knowledge to enable: The application of genomic technology in microbiology research - assignment °E.coli systems ML °Bacteria other than E. coliJQ °PathogensSP

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Overview of diversity Environmental Influence °DNA Replication °Mutation °Repair T o C, pH, eH salinity, growth rate, UV radiation, chemical damage °Recombination °Homologous °Non-homologous °Rearrangements °Deletions DNA Transformation Phage Transduction Plasmid Conjugation

©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.

©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

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Basic mechanisms/models Breaking and Joining of DNA –Strand exchange. RECOMBINATION Types. –Homologous / general –Non-homologous / illegitimate –Site specific –Replicative / transposition Breaking and Joining of DNA –Strand exchange. RECOMBINATION Types. –Homologous / general –Non-homologous / illegitimate –Site specific –Replicative / transposition HOMOLOGOUS RECOMBINATION Reciprocal exchange Gene conversion / segregation replication not required THEORIES Chiasmatic Janssens 1909 Darlington 1930 Copy ChoiceBelling 1931 Sturtevant 1949 Lederberg 1955

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Recombination in bacteria Recombination in Transformation Invasion by Single stranded DNA Recombination in Transduction/ Transfection Invasion by Double stranded DNA (Mismatch repair) Recombination in conjugation Transfer and invasion by Single stranded DNA Recombination between intact Double stranded molecules

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Recombination in conjugation Single Stranded DNA Transferred Evidence e.g.. MateHfr WT ( i.e. LacZ + ) X F - lacZ Mutant WT LacZ Single stranded DNA Mutated LacZ mRNA Defective  -galactosidase mRNA Functioning  -galactosidase WT - Invasion recA - NO invasion recBCD - Invasion

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Holliday Model 3’ 5’ A T C G 3’ 5’ A T C G 3’ 5’ T C G A C A T G BRANCH MIGRATION C A G T A C T G Chi -intermediate

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Holliday Model contd Endonuclease Nicking Resolve OR T G A C A C T G C A G T C A G T

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Isomerisation of a 1-strand X-over to produce a 2-strand X-over. OR 3’end5’end OR

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. What happens to mismatched pairs after recombination? Gene conversion. DNA Replication Post-meiotic segregation A C A C A T G C A C Excision A T G C Repair A C Mismatch repair

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Meselson- Radding Model for Bacterial Recombination 1. Single strand nick 2. Dissociation of ssDNA 3. Assimilation into D-Loop 4. D-Loop Digestion 5. Ligation 6. Branch migration 7. Complete Recombination

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Recombination between Replicons. Chi- intermediates Valenzuela & Inman ( -Phage) Potter and Dressler (ColE1 Plasmid) ColE1 amplified in E.coli by chloramphenicol. up to 1000 copies. Many recombine by homologous recombination Extract and examine under EM - high magnification Either INTERLOCKING circles OR Recombinational intermediates

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Demonstration of Chi- intermediates. Cut ColE1 at single EcoR1 site. Agarose gel and Et Br stain A B All Linear DNA ? OR Some still Joined ? Observe using EM EcoR1 Cuts TWIST Chi

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Resolution of Chi-intermediates. Role of Endonucleases Cuts 2 and 4 Cuts 1 and 3 Cuts 1 and 2 or 3 and 4

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. Development of Recombination Models “The prudent Scientist keeps his (sic) hypothesis simple. He endows it with only enough complexity to account for his observations. He stands stubbornly by his simple observations; resisting the complicating results of other people’s experiments, until there is no further doubt of their validity. Then he retreats a very short way, taking up a new position; with only enough added complexity to accommodate the unwanted findings. There he digs in and prepares for the next attack of the anarchist.” D. Stadler (1973) Annual Review of Genetics Vol7.

©M J Larkin Biology & Biochemistry. The Queen’s University of Belfast. The question remains the same! What is the role of homologous recombination in the evolution of microbial genomes? 1973 – a detailed answer possible – but mechanisms not known and significance of non- homologous recombination uncertain 2006 – mechanisms better resolved – must now discuss in relation to transposons and other mechanisms. Whole genomes data confuse the picture!