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Lambda RED Recombination

Lambda RED Recombination. JIC in Norwich. University of Tübingen. Genomic organisation of the lambdoid bacteriophage. Genes exo , bet and gam are clustered in the P L operon.

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Lambda RED Recombination

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  1. Lambda RED Recombination JIC in Norwich University of Tübingen

  2. Genomic organisation of the lambdoid bacteriophage Genes exo, bet and gam are clustered in the PL operon. red- (recombination defective)  mutants were partially defective in homologous recombination in a wt-host, and grossly defective in a recA- host. The E. coli recombination system primarily restores collapsed replication forks, repairs DSB and maintains the genetic integrity of the E. coli chromosome. During the replication of , the infected cell is a hotbed of genetic exchange (hyper-rec state).

  3. How does  Red stimulate homologous recombination ? linear DNA 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ circular DNA

  4.  Gam () inhibits the exonuclease V activity of the recBCD system linear DNA   Rec BCD Rec BCD 3´ 5´   5´ 3´ 3´ 5´ 5´ 3´ circular DNA  Gam () binds as a dimer to the E. coli RecBCD complex and inhibits its nuclease activity

  5.  Exo () binds to dsDNA ends ... linear DNA   3´ 5´ 5´ 3´ Subramanian et al., 2003 3´ 5´ 5´ 3´ circular DNA •  Exo () degrades linear dsDNA in 5´ to 3´direction (1kb/sec in-vitro) leaving long 3´ssDNA overhangs • The active form of the protein (24kDa) is a trimer with a central hole

  6. ... and progressively generates 3´ overhangs linear DNA   3´ 3´ Subramanian et al., 2003 3´ 5´ 5´ 3´ circular DNA The entrance of the hole accommodates dsDNA, the exit diameter is the size of ssDNA

  7.  Beta (ß) binds to ssDNA and mediates invasion of the ssDNA into an unbroken homologous duplex linear DNA 3´ ß ß ß ß ß ß ß ß 3´ 3´ 5´ 5´ 3´ circular DNA •  Beta (ß) binds to ssDNA greater than 35 nucleotides in length • Beta belongs to a family of recombination proteins which include Erf protein of Salmonella phage P22, the RecT protein of the cryptic E. coli phage Rac, and the Rad52 protein of eukaryotes • Beta promotes denaturation of complementary strands, strand annealing and exchange reactions

  8. RecFOR is essential for the formation of the recombination complex linear DNA RecFOR 3´ 5´ ß ß ß ß 3´ ß ß ß ß 3´ 5´ 3´ circular DNA RecFOR • RecFOR replaces single-strand binding proteins (SSB) bound on ssDNA with RecA • RecA stabilises complex of Bet, DNA and RecFOR

  9. RuvAB helicase-driven branch migration results in Holliday junction formation ... linear DNA Rafferty et al., 1996 RuvAB 3´ 5´ ß ß ß ß 3´ ß ß ß ß 3´ 5´ 3´ circular DNA RuvAB RuvAB recognizes a four-way junction ( Holliday junction) and catalyzes branch migration

  10. ... which can be resolved by RuvC ... linear DNA RuvC RuvC Rafferty et al., 1996 3´ 5´ ß ß ß ß 3´ ß ß ß ß 3´ 5´ 3´ circular DNA RuvC is a Holliday junction endonuclease (structure specific resolvase)

  11. ... into a recombinant molecule 3´ 5´ 5´ 3´

  12. PCRproduct FRT FRT marker oriT P1 P2 39bp 39bp X X FRT FRT marker oriT P1 P2 S. coelicolor cosmid neo bla PCR-targeting (step 1) target S. coelicolor cosmid neo bla SuperCos1

  13. FRT FRT marker X X oriT P1 P2 S. coelicolor cosmid FRT FRT marker oriT P1 P2 neo bla SuperCos1 S. coelicolor chromosome PCR-targeting (step 2) S. coelicolor chromosome target

  14. Why two-step strategy?  Red is efficient in E. coli Recombinant cosmid-DNA can easily be confirmed by PCR, restriction analysis and/or sequencing Mutagenised cosmids can be mobilised by conjugation, no need for transformation procedures High frequency of double cross-overs due to long flanking sequences in a cosmid clone Disadvantages Dependent upon the availability of a E. coli clone Not high throughput (in comparison to transposon mutagenesis)

  15. NEW FRT FRT hyg pIJ797 oriT P1 P2 Template cassettes for gene replacements FRT FRT FRT FRT vph aac(3)IV pIJ773 pIJ780 oriT P1 P2 oriT P1 P2 FRT FRT loxP loxP vph vph aac(3)IV pIJ781 pIJ774 P1 P2 oriT P1 P2 SwaI SwaI FRT FRT tet aac(3)IV pIJ782 pIJ775 oriT P1 P2 P2 oriT P1 FRT FRT neo pIJ776 oriT P1 P2 FRT FRT neo pIJ777 P1 P2 FRT FRT aadA pIJ778 oriT P1 P2 FRT FRT aadA pIJ779 P1 P2

  16. FRT FRT aac(3)IV pIJ785 oriT tipAp P2 P1 FRT FRT aac(3)IV pMS80 P2 oriT tcp P1 FRT FRT aac(3)IV NEW NEW NEW oriT fd-ter P1 P2 nitAp Template cassettes for other applications int tet aac(3)IV pIJ787 pIJ784 oriT bla bla attP oriT bla bla hyg pIJ798 oriT bla bla aac(3)IV pIJ789 neo neo aac(3)IV pIJ794 oriT neo neo vph pIJ795 oriT neo neo FRT FRT aac(3)IV egfp pIJ786 oriT aadA P1 P2 pIJ796 neo oriT neo S. coelicolor cosmid neo bla Herai et al., 2004. Hyper-inducible expression system for streptomycetes. PNAS101, 14031-14035

  17. REDIRECT (Rapid Efficient Directed Recombination Time saving)Download protocol and Primer design program at http://streptomyces.org.uk/redirect/index.htmlTo obtain the REDIRECT KIT: Mail Nicholas Bird nicholas.bird@bbsrc.ac.uk USA 26 United Kingdom 21 Germany 12 Canada 7 Spain 7 China 5 Korea 4 Netherlands 2 Japan 2 Taiwan 2 Argentina 1 Belgium 1 Finland 1 France 1 Israel 1 Sweden 1 Switzerland 1 Taiwan 1 Gust, B., Chandra, G., Jakimowicz, D., Tian, Y., Bruton, C.J. and Chater, K.F. (2004) λ Red-mediated genetic manipulation of antibiotic-producing Streptomyces, Advances in Applied Microbiology54: 107-28. Gust, B. Challis, G.L., Fowler, K., Kieser, T. and Chater, K.F. (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odour geosmin, Proc. Natl. Acad. Sci.USA100: 1541-6 Gust, B., Kieser, T. and Chater, K.F. (2002) REDIRECT technology: PCR targeting system in Streptomyces coelicolor, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom

  18. Epitope tagging using REDIRECT a b 1. PCR ab FRT FRT aac(3)IV P1 P2 oriT c FRT FRT aac(3)IV 2. PCR ac Tag P1 P2 oriT cosmid neo bla

  19. Epitope tagging using REDIRECT a b 1. PCR ab FRT FRT aac(3)IV P1 P2 oriT c FRT FRT aac(3)IV 2. PCR ac Tag P1 P2 oriT cosmid neo bla

  20. Introducing point mutations using REDIRECT aac(3)IV pIJ775 oriT P1 P2 SwaI I-SceI SwaI 40 bp 40 bp * * PCR-product X X P1 P2 cosmid neo bla select for KanR CarbR transformants

  21. leading 5´ DnaB DNA Pol III 3´ 3´ 5´ 5´ 5´ lagging Single strand oligonucleotide repair (ssOR) Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cellsYouming Zhang, Josep PP Muyrers, Jeanette Rientjies and A. Francis StewardBMC Molecular Biology 2003, 4:1-14 • Only λ Bet is required • Strand bias: more ssOR with oligos priming the lagging strand • Efficiency of ssOR is maximum with oligos ~ 120 nt

  22. 120bp dsDNA Afl II S. coelicolor cosmid neo bla Oligo-Targeting for deleting transposon insertions Tn5062 S. coelicolor cosmid neo bla

  23. 120bp dsDNA I-SceI S. coelicolor cosmid neo bla Oligo-Targeting for generation of “scar less” in-frame deletions Cyc2 aac(3)IV P2 oriT P1 S. coelicolor cosmid neo bla

  24. Summary

  25. Acknowledgements Department of Molecular Microbiology Celia Bruton Prof. Keith Chater Sir David Hopwood Tobias Kieser Helen Kieser Greg Challis Kay Fowler Prof. Barry Wanner Prof. Mervyn Bibb Mark Buttner

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