Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate.

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.

Introduction.
Red Lambda system.
Powerful technique for making: Insertions. Deletions. Points mutations. Chromosomal targets. BACs. Plasmids.

This system has been used to modify:

Introduction.
Red Lambda system.
Proteins: Gam. Exo. Beta.
RecBCD SbcCD

METHODS.
E.coli EcNR2

dsDNA

Electroporation.

dsDNA/phosphorothioato

Recombineering

dsDNA/mismatch MAMA-PCR

Lambda Red-mediated recombination mechanisms.

dsDNA

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).

Lambda Red-mediated recombination mechanisms.

dsDNA

Figure 1.Previously proposed lambda Red-mediated dsDNA recombination mechanisms. (B) The Poteete mechanism suggests that (1) Beta facilitates 3 overhang annealing to the lagging strand of the replication fork and (2) positions the invading strand to serve as the new template for leading-strand synthesis. This structure is resolved by an unspecified host endonuclease (red triangle), and (3) the synthetic dsDNA becomes template for both lagging and leadingstrand synthesis. A second template switch must then occur at the other end of the synthetic dsDNA (Poteete 2008).

Lambda Red mediated recombination proceeds via a intermediate.

dsDNA ssDNA

Figure 2.Lambda Red mediated dsDNA recombination proceeds via a ssDNA intermediate. Instead of a recombination intermediate involving dsDNA flanked by 3ssDNA 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 for gene replacement (as shown) or for insertions in which no genomic DNA is removed.

Strand bias in lambda Red ssDNA insertion recombination.

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 leadingtargeting strands. An asterisk indicates P , 0.05.

Strand-specific mismatch alleles were used to identify the strand of origin for each recombined mutation.

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

Tracking cosegregation in mismatched dsDNA recombination.

Testing the effect of recombination frequency.

strand

protection

on

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.

Thanks.

INFO.
Consegregacion: Transmisin conjunta de dos o ms genes ligados en un mismo cromosoma.