7/30/2019 Lambda RED Recombineering

1/13

Lambda Red Recombineering in Escherichia coliOccurs Through a Fully Single-StrandedIntermediate.J. A. Mosberg,*,,1,2 M. J. Lajoie*,,1,2 and G. M. Church*

*Department of Genetics, Harvard Medical School, Boston, Massachusetts

02115 and Program in Chemical Biology, Harvard University, Cambridge,

Massachusetts 02138.

Javier Villacreses.

7/30/2019 Lambda RED Recombineering

2/13

Introduction.

Red Lambda system.

Powerful technique for making: Insertions.Deletions.

Points mutations.

This system has been used to modify: Chromosomal targets.

BACs.

Plasmids.

7/30/2019 Lambda RED Recombineering

3/13

Introduction.

Red Lambda system.

Proteins: Gam.

Exo.

Beta.

RecBCD

SbcCD

7/30/2019 Lambda RED Recombineering

4/13

METHODS.

E.coli

MAMA-PCR

EcNR2

Recombineering

dsDNAElectroporation.

dsDNA/phosphorothioato

dsDNA/mismatch

7/30/2019 Lambda RED Recombineering

5/13

Lambda Red-mediated dsDNArecombination mechanisms.

Figure 1.Previously proposed lambda Red-mediated dsDNA recombination mechanisms. Heterologous dsDNA

is shown in green; Exo is an orange oval, and Beta is a yellow oval. In both mechanisms the recombination

intermediate is proposed to be a dsDNA core flanked on either side by 3 ssDNA overhangs. (A) The Court

mechanism posits that (1) Beta facilitates annealing of one 3 overhang to the lagging strand of the replication fork.

(2) This replication fork then stalls and backtracks so that the leading strand can template switch onto the

synthetic dsDNA. The heterologous dsDNA blocks further replication from this fork. (3) Once the second

replication fork reaches the stalled fork, the other 3 end of the integration cassette is annealed to the lagging

strand in the same manner as prior. Finally, the crossover junctions must be re- solved by unspecified E. coli

enzymes (Court et al. 2002).

7/30/2019 Lambda RED Recombineering

6/13

7/30/2019 Lambda RED Recombineering

7/13

Lambda Red mediated dsDNArecombination proceeds via a ssDNAintermediate.

Figure 2.Lambda Red mediated dsDNA recombination proceeds via a ssDNA

intermediate. Instead of a recombination intermediate involving dsDNA flanked by 3-

ssDNA overhangs, we propose that one strand of linear dsDNA is entirely degraded

by Exo (orange oval). Beta (yellow oval) then facilitates annealing to the lagging

strand of the replication fork in place of an Okazaki fragment. The heterologous

region does not anneal to the genomic sequence. This mechanism could account forgene replacement (as shown) or for insertions in which no genomic DNA is removed.

7/30/2019 Lambda RED Recombineering

8/13

Strand bias in lambda Red ssDNA insertionrecombination.

Figure 3.Strand bias in lambda

Red ssDNA insertion

recombination. Recombination

frequencies were assessed for

several leading-targeting and

lagging-targeting complementary

ssDNA pairs. Lagging-targeting

strands were found to be more

recombinogenic than leading-

targeting strands. An asterisk

indicates P , 0.05.

7/30/2019 Lambda RED Recombineering

9/13

Strand-specific mismatch alleles were used toidentify the strand of origin for each recombinedmutation.

Figure 4.Strand-specific mismatch alleles were used to identify the strand of origin for each recombined mutation. The mismatchedlacZ::kanR cassette contained two consecutive mismatches at two loci in both flanking homology regions. Strand 1 was the lagging-

targeting strand and strand 2 was the leading-targeting strand. If lambda Red dsDNA recombination proceeds via a ssDNA

intermediate (left), (a) one Exo (orange oval) binds to a dsDNA end, (b) Exo fully degrades one strand while helping to load Beta

(yellow oval) onto the remaining strand, and (c) this strand provides all of the genetic information during recombination. This figure

shows the case in which the lagging-targeting strand is recombined (coding-strand genotypes: L1, AA; L2, AA; L3, TT; L4, TT), but

the leading-targeting strand is also predicted to be observed (coding-strand genotypes: L1, CC; L2, CC; L3, GG; L4, GG). If the

lambda Red recombination intermediate is a heterologous dsDNA core flanked by 3 -ssDNA overhangs (right), (a) one Exo binds to

each dsDNA end, (b) Exo recesses both strands while helping to load Beta onto both 3 overhangs, and (c) both strands provide

genetic information for each recombination. Since Exo always degrades 5 3, the expected coding-strand genotypes for the Court

and Poteete mechanisms would be L1, CC; L2, CC; L3, TT; L4, TT.

7/30/2019 Lambda RED Recombineering

10/13

Tracking cosegregation in mismatched

dsDNA recombination.

7/30/2019 Lambda RED Recombineering

11/13

Testing the effect of strand protection on

recombination frequency.

Figure 5. Testing the effect of strand protection on recombination frequency. Four lacZ::kanR cassettes were tested to

determine whether protecting one strand has a greater effect on recombination frequency than protecting the other strand.

In each case, protection was accomplished through the placement of four phosphorothioate linkages on the 5 end of a

strand. Inset: Analysis of variance for lagging-targeting (Lag) phosphorothioation and leading-targeting (Lead)

phosphorothioation. An asterisk (*) denotes phosphorothioation. Lagging-targeting phosphorothioation was found to

significantly enhance recombination frequency, whereas leading-targeting phosphorothioation did not affect recombination

frequency.

7/30/2019 Lambda RED Recombineering

12/13Thanks.

7/30/2019 Lambda RED Recombineering

13/13

Consegregacion: Transmisin conjunta de dos o ms genes ligados en

un mismo cromosoma.

INFO.