Lambda RED Recombination : Lambda RED Recombination JIC in Norwich University of Tübingen
Genomic organisation of the lambdoid bacteriophage : 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).
How does Red stimulate homologous recombination ? : How does Red stimulate homologous recombination ? 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ circular DNA linear DNA
Gam () inhibits the exonuclease V activity of the recBCD system : Gam () inhibits the exonuclease V activity of the recBCD system 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ circular DNA linear DNA Rec
BCD Rec
BCD Gam () binds as a dimer to the E. coli RecBCD complex and inhibits its nuclease activity
Exo () binds to dsDNA ends ... : Exo () binds to dsDNA ends ... 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ circular DNA linear DNA
Subramanian et al., 2003 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
... and progressively generates 3´ overhangs : ... and progressively generates 3´ overhangs 3´ 3´ 3´ 5´ 5´ 3´ circular DNA linear DNA
The entrance of the hole accommodates dsDNA, the exit diameter is the size of ssDNA Subramanian et al., 2003
Beta (ß) binds to ssDNA and mediates invasion of the ssDNA into an unbroken homologous duplex : Beta (ß) binds to ssDNA and mediates invasion of the ssDNA into an unbroken homologous duplex 3´ 3´ 3´ 5´ 5´ 3´ circular DNA linear DNA ß ß ß ß 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 ß ß ß ß
RecFOR is essential for the formation of the recombination complex : RecFOR is essential for the formation of the recombination complex 3´ 3´ RecFOR RecFOR 3´ 5´ 5´ 3´ RecFOR replaces single-strand binding proteins (SSB) bound on ssDNA with RecA
RecA stabilises complex of Bet, DNA and RecFOR circular DNA linear DNA ß ß ß ß ß ß ß ß
RuvAB helicase-driven branch migration results in Holliday junction formation ... : RuvAB helicase-driven branch migration results in Holliday junction formation ... 3´ 3´ RuvAB RuvAB 3´ 5´ 5´ 3´ Rafferty et al., 1996 circular DNA linear DNA ß ß ß ß ß ß ß ß RuvAB recognizes a four-way junction ( Holliday junction) and catalyzes branch migration
... which can be resolved by RuvC ... : ... which can be resolved by RuvC ... 3´ 3´ RuvC RuvC 3´ 5´ 5´ 3´ RuvC is a Holliday junction endonuclease (structure specific resolvase)
circular DNA linear DNA Rafferty et al., 1996 ß ß ß ß ß ß ß ß
... into a recombinant molecule : ... into a recombinant molecule 3´ 5´ 5´ 3´
PCR-targeting (step 1) : PCR-targeting (step 1) S. coelicolor cosmid neo SuperCos1 bla target
PCR-targeting (step 2) : PCR-targeting (step 2) target S. coelicolor chromosome
Why two-step strategy? : 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)
Template cassettes for gene replacements : Template cassettes for gene replacements P1 P2 aac(3)IV oriT FRT FRT P1 P2 aac(3)IV oriT loxP loxP P1 P2 oriT aac(3)IV SwaI SwaI pIJ773 pIJ774 pIJ775 P1 P2 aadA oriT FRT FRT P1 P2 neo oriT P1 P2 aadA FRT FRT P1 P2 neo FRT FRT pIJ776 pIJ777 pIJ778 pIJ779 FRT FRT P1 P2 vph oriT FRT FRT P1 P2 vph FRT FRT pIJ780 pIJ781 vph P1 P2 tet oriT FRT FRT pIJ782
Template cassettes for other applications : Template cassettes for other applications attP bla bla tet int oriT pIJ787 Herai et al., 2004. Hyper-inducible expression system for streptomycetes. PNAS 101, 14031-14035 P1 P2 aac(3)IV oriT FRT FRT egfp pIJ786 bla bla aac(3)IV oriT pIJ784 bla bla hyg oriT pIJ798 neo neo aac(3)IV aac(3)IV pIJ789 pIJ794 oriT neo neo neo neo neo neo oriT oriT aadA vph pIJ795 pIJ796 S. coelicolor cosmid neo bla
REDIRECT (Rapid Efficient Directed Recombination Time saving) Download protocol and Primer design program at http://streptomyces.org.uk/redirect/index.html To obtain the REDIRECT KIT: Mail Nicholas Bird nicholas.bird@bbsrc.ac.uk : REDIRECT (Rapid Efficient Directed Recombination Time saving) Download protocol and Primer design program at http://streptomyces.org.uk/redirect/index.html To obtain the REDIRECT KIT: Mail Nicholas Bird nicholas.bird@bbsrc.ac.uk 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 Microbiology 54: 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. USA 100: 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 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
Epitope tagging using REDIRECT : Epitope tagging using REDIRECT P1 P2 FRT FRT oriT aac(3)IV P1 P2 FRT FRT oriT aac(3)IV a b c 1. PCR ab 2. PCR ac Tag cosmid neo bla
Epitope tagging using REDIRECT : cosmid Epitope tagging using REDIRECT P1 P2 FRT FRT oriT aac(3)IV P1 P2 FRT FRT oriT aac(3)IV a b c 1. PCR ab 2. PCR ac Tag neo bla
Introducing point mutations using REDIRECT : cosmid neo bla P1 P2 PCR-product 40 bp 40 bp X X select for KanR CarbR transformants * * P1 P2 pIJ775 SwaI I-SceI SwaI oriT aac(3)IV Introducing point mutations using REDIRECT
Single strand oligonucleotide repair (ssOR) : Single strand oligonucleotide repair (ssOR) Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells Youming Zhang, Josep PP Muyrers, Jeanette Rientjies and A. Francis Steward BMC 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
Oligo-Targeting for deleting transposon insertions : Oligo-Targeting for deleting transposon insertions S. coelicolor cosmid neo bla Tn5062
Oligo-Targeting for generation of “scar less” in-frame deletions : Oligo-Targeting for generation of “scar less” in-frame deletions S. coelicolor cosmid neo bla Cyc2 P1 P2 oriT aac(3)IV
Summary : Summary
Acknowledgements : Acknowledgements Department of Molecular Microbiology Prof. Keith Chater Tobias Kieser Helen Kieser Celia Bruton Kay Fowler Greg Challis Sir David Hopwood Prof. Mervyn Bibb Mark Buttner Prof. Barry Wanner