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

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Lambda Red Recombineering in Escherichia Coli Occurs Through a Fully Single-Stranded Intermediate Copyright Ó 2010 by the Genetics Society of America DOI: 10.1534/genetics.110.120782 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 Manuscript received July 12, 2010 Accepted for publication August 30, 2010 ABSTRACT The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 59 ends of double-stranded DNA are recessed by l exonuclease, leaving behind 39 overhangs. Here, we propose an alternative in which lambda exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA. This single-stranded intermediate then recombines via beta recombinase- catalyzed annealing at the replication fork. We support this by showing that single-stranded gene insertion cassettes are recombinogenic and that these cassettes preferentially target the lagging strand during DNA replication. Furthermore, a double-stranded DNA cassette containing multiple internal mismatches shows strand-specific mutations cosegregating roughly 80% of the time. These observations are more consistent with our model than with previously proposed models. Finally, by using phosphorothioate linkages to protect the lagging-targeting strand of a double-stranded DNA cassette, we illustrate how our new mechanistic knowledge can be used to enhance lambda Red recombination frequency. The mechanistic insights revealed by this work may facilitate further improvements to the versatility of lambda Red recombination. VER the past decade, lambda Red recombination 2000), and to develop novel cloning methods (Lee O (‘‘recombineering’’) has been used as a power- et al. 2001; Li and Elledge 2005). Large-scale dsDNA ful technique for making precisely defined insertions, recombineering projects include creating a library of deletions, and point mutations in Escherichia coli, single-gene knockout E. coli strains (Baba et al. 2006) requiring as few as 35 bp of homology on each side and removing 15% of the genomic material from a of the desired alteration (Thomason et al. 2007a; single E. coli strain (Posfai et al. 2006). Linear dsDNA Sharan et al. 2009). With this system, single-stranded recombineering has also been used to insert heterol- DNA (ssDNA) oligonucleotides have been used to ogous genes and entire pathways into the E. coli efficiently modify E. coli chromosomal targets (Ellis chromosome (Zhang et al. 1998; Wang and Pfeifer et al. 2001; Costantino and Court 2003), BACs 2008) and BACs (Lee et al. 2001; Warming et al. 2005), (Swaminathan et al. 2001), and plasmids (Thomason including those used for downstream applications in et al. 2007b), as well as to rapidly optimize a metabolic eukaryotes (Chaveroche et al. 2000; Bouvier and pathway coding for the production of lycopene (Wang Cheng 2009). However, despite the broad use of this et al. 2009). Furthermore, linear double-stranded DNA method, the mechanism of lambda Red recombination (dsDNA) recombineering has been used to replace has not achieved scientific consensus, particularly in chromosomal genes (Murphy 1998; Murphy et al. 2000), the case of dsDNA recombination. A clearer under- to disrupt gene function (Datsenko and Wanner standing of the mechanism underlying this process could suggest ways to improve the functionality, ease, and versatility of lambda Red recombination. Supporting information is available online at http://www.genetics.org/ cgi/content/full/genetics.110.120782/DC1. Three phage-derived lambda Red proteins are neces- Available freely online through the author-supported open access sary for carrying out dsDNA recombination: Gam, Exo, option. and Beta. Gam prevents the degradation of linear 1These authors contributed equally to this work. dsDNA by the E. coli RecBCD and SbcCD nucleases; 2Corresponding authors: Department of Genetics, Harvard Medical lambda exonuclease (Exo) degrades dsDNA in a 59 to School, 77 Avenue Louis Pasteur, New Research Bldg., Room 238, Boston, MA 02115. E-mail: [email protected] and mlajoie@ 39 manner, leaving single-stranded DNA in the recessed fas.harvard.edu regions; and Beta binds to the single-stranded regions Genetics 186: 791–799 (November 2010) 792 J. A. Mosberg, M. J. Lajoie and G. M. Church produced by Exo and facilitates recombination by promoting annealing to the homologous genomic target site (Sawitzke et al. 2007). Current mechanisms claim that Exo binds to both 59 ends of the dsDNA and degrades in both directions simultaneously to produce a double-stranded region flanked on both sides by 39 overhangs (Sharan et al. 2009; Szczepanska 2009). However, a comprehensive explanation of how this construct ultimately recombines with the chromosome has not yet been advanced. Initially, it was proposed that this recombination occurs via strand invasion (Thaler et al. 1987). How- ever, it has more recently been shown that strand invasion is unlikely to be the dominant mechanism in the absence of long regions of homology, as recombi- nation remains highly proficient in a recA- background (Yu et al. 2000). Furthermore, a detailed analysis of lambda Red recombination products showed character- istics consistent with strand annealing rather than a strand invasion model (Stahl et al. 1997). Finally, lambda Red dsDNA recombination has been shown to preferentially target the lagging strand during DNA Figure 1.—Previously proposed lambda Red-mediated replication, which suggests strand annealing rather dsDNA recombination mechanisms. Heterologous dsDNA is than strand invasion (Lim et al. 2008; Poteete 2008). shown in green; Exo is an orange oval, and Beta is a yellow oval. To explain these results, Court et al. (2002) proposed In both mechanisms the recombination intermediate is pro- 9 a strand-annealing model for insertional dsDNA re- posed to be a dsDNA core flanked on either side by 3 ssDNA overhangs. (A) The Court mechanism posits that (1) Beta fa- combination (Figure 1A), in which one single-stranded cilitates annealing of one 39 overhang to the lagging strand 39 end anneals to its homologous target at the replica- of the replication fork. (2) This replication fork then stalls tion fork. The replication fork then stalls, due to the and backtracks so that the leading strand can template switch presence of a large dsDNA nonhomology (i.e., the onto the synthetic dsDNA. The heterologous dsDNA blocks insertion cassette). The stalled replication fork is ulti- further replication from this fork. (3) Once the second repli- cation fork reaches the stalled fork, the other 39 end of the in- mately rescued by the other replication fork traveling tegration cassette is annealed to the lagging strand in the same in the opposite direction around the circular bacterial manner as prior. Finally, the crossover junctions must be re- chromosome. The other 39 end of the recombinogenic solved by unspecified E. coli enzymes (Court et al. 2002). DNA anneals to the homology region exposed by the (B) The Poteete mechanism suggests that (1) Beta facilitates 9 second replication fork, forming a crossover structure, 3 overhang annealing to the lagging strand of the replication fork and (2) positions the invading strand to serve as the new which is then resolved by unspecified E. coli enzymes template for leading-strand synthesis. This structure is resolved (Court et al. 2002). by an unspecified host endonuclease (red triangle), and (3) The Court mechanism was challenged by Poteete the synthetic dsDNA becomes template for both lagging and leading-strand synthesis. A second template switch must then (2008), who showed that the dsDNA recombination of oteete a linear lambda phage chromosome occurs readily onto occur at the other end of the synthetic dsDNA (P 2008). The figure was adapted from the references cited. a unidirectionally replicating plasmid, which does not have the second replication fork required by the Court mechanism (Court et al. 2002). Thus, Poteete pro- While Poteete’s mechanism addresses some of the posed an alternate mechanism (Poteete 2008), termed weaknesses of the Court mechanism, it remains largely ‘‘replisome invasion’’(Figure 1B), in which a 39 overhang speculative. This mechanism does not identify the of the Exo-processed dsDNA first anneals to its comple- endonuclease responsible for resolving the structure mentary sequence on the lagging strand of the recom- after the first template switching event, nor does it bination target. Subsequently, this overhang displaces explain how the recombinogenic DNA and replication the leading strand, thereby serving as the new template machinery form a new replication fork. Additionally, for leading-strand synthesis. The resulting structure is this template-switching mechanism would have to oper- resolved by an unspecified endonuclease, after which ate two times in a well-controlled manner, which may the recombinogenic DNA becomes the template for the not be consistent with the high-recombination frequen- synthesis of both new strands. In
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