Lambda RED Recombination

11.07.2005 Bertolt Gust

Lambda RED Recombination

JIC in Norwich University of Tübingen


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 ?

5´

3´

3´

5´

3´

5´

5´

3´

circular DNA

linear DNA


Gam () inhibits the exonuclease V activity of the recBCD system

5´

3´

3´

5´

3´

5´

5´

3´

circular DNA

linear DNA

RecBCD

RecBCD

Gam () binds as a dimer to the E. coli RecBCD complex and inhibits its nuclease activity


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

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

3´

3´

3´

5´

5´

3´

circular DNA

linear DNA

ß ßß 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 ßß ß


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 ßß ß

ß ßß ß


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 ßß ß

ß ßß ß

RuvAB recognizes a four-way junction ( Holliday junction) and catalyzes branch migration


... 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 ßß ß

ß ßß ß


... into a recombinant molecule

3´

5´

5´

3´


PCR-targeting (step 1)

S. coelicolor cosmid

neoSuperCos1

bla

targetX X39bp 39bpP1 P2markeroriT

FR

T

FR

T PCR product


neo bla

P1 P2markeroriT

FR

T

FR

T


PCR-targeting (step 2)

targetS. coelicolor chromosome


neoSuperCos1

bla

X XP1 P2markeroriT

FR

T

FR

T

P1 P2markeroriT

FR

T

FR

T

S. coelicolor chromosome


Why two-step strategy?

1. Red is efficient in E. coli

2. Recombinant cosmid-DNA can easily be confirmed by PCR, restriction analysis and/or sequencing

3. Mutagenised cosmids can be mobilised by conjugation, no need for transformation procedures

4. High frequency of double cross-overs due to long flanking sequences in a cosmid clone

Disadvantages

1. Dependent upon the availability of a E. coli clone

2. Not high throughput (in comparison to transposon mutagenesis)


Template cassettes for gene replacements

P1 P2aac(3)IVoriT

FR

T

FR

T

P1 P2aac(3)IVoriT

loxP

loxP

P1 P2oriTaac(3)IV

SwaI SwaI

pIJ773

pIJ774

pIJ775

P1 P2aadAoriT

FR

T

FR

T

P1 P2neooriT

P1 P2aadA

FR

T

FR

T

P1 P2neo

FR

T

FR

T

pIJ776

pIJ777

pIJ778

pIJ779

FR

T

FR

T

P1 P2vphoriT

FR

T

FR

T

P1 P2vph

FR

T

FR

T

pIJ780

pIJ781 vph

P1 P2tetoriTFR

T

FR

TpIJ782

P1 P2hygoriT

FR

T

FR

TpIJ797 NEW


Template cassettes for other applications

attPbla blatet intoriTpIJ787

P1 tipApP2oriT

FR

T

FR

Taac(3)IVpIJ785

P1 tcpP2oriT

FR

T

FR

Taac(3)IVpMS80

P1 P2aac(3)IVoriT

FR

T

FR

T nitApfd-terNEW

Herai et al., 2004. Hyper-inducible expression system for streptomycetes. PNAS 101, 14031-14035

P1 P2aac(3)IVoriT

FR

T

FR

TegfppIJ786

bla blaaac(3)IVoriTpIJ784

bla blahygoriTpIJ798 NEW

neo neoaac(3)IV

aac(3)IV

pIJ789

pIJ794 oriTneo neo

neo neo

neo neooriT

oriT

aadA

vphpIJ795

pIJ796NEW

S. coelicolor cosmidneo 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 [email protected]

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 26United Kingdom 21Germany 12Canada 7Spain 7China 5Korea 4Netherlands 2Japan 2Taiwan 2Argentina 1Belgium 1Finland 1France 1Israel 1Sweden 1Switzerland 1Taiwan 1


Epitope tagging using REDIRECT

P1 P2

FR

T

FR

ToriTaac(3)IV

P1 P2

FR

T

FR

ToriTaac(3)IV

ab

c

1. PCR ab

2. PCR acTag

cosmid

neo bla


cosmid

Epitope tagging using REDIRECT

P1 P2

FR

T

FR

ToriTaac(3)IV

P1 P2

FR

T

FR

ToriTaac(3)IV

ab

c

1. PCR ab

2. PCR acTag

neo bla


cosmidcosmid

neo bla

P1 P2

PCR-product

40 bp 40 bp

X X

select for KanR CarbR transformants

* ** *

P1 P2pIJ775

SwaI I-SceI SwaI

oriTaac(3)IV

Introducing point mutations using REDIRECT


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

DNA Pol IIIDnaB

5´3´

5´

5´3´

5´

leading

lagging


Oligo-Targeting for deleting transposon insertions


neo bla

Tn5062

120bp dsDNA


neo bla

Afl Afl IIII


Oligo-Targeting for generation of “scar less” in-frame deletions


neo bla

Cyc2

120bp dsDNA


neo bla

P1 P2oriTaac(3)IV

I-SceI


Summary

PCR-targeting Oligo-targeting

gene knock-outs gene replacements generating point mutations

gene fusions module swapping inserting restriction sites

epitope tagging inserting point mutations

“scarless” mutations

promoter replacements ET-cloning N-terminal epitope tagging

integration of cosmids (attP)

promoter replacements deletion of transposon insertion


Acknowledgements

Department of Molecular Microbiology

Prof. Keith Chater Tobias Kieser Helen Kieser

Celia Bruton

Kay FowlerGreg Challis

Sir David Hopwood

Prof. Mervyn Bibb Mark Buttner Prof. Barry Wanner

Documents

Lambda RED Recombination