Lambda Red Recombineering in Escherichia Coli Occurs Through a Fully Single Stranded Intermediate
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Genetics: Published Articles Ahead of Print, published on September 2, 2010 as 10.1534/genetics.110.120782 Lambda Red Recombineering in Escherichia coli occurs through a fully singlestranded intermediate Mosberg, J. A.* §, Lajoie, M. J.* §, and Church, G. M. * These authors contributed equally to this work. Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, § Program in Chemical Biology, Harvard University, Cambridge, Massachusetts 02138 1 Running Title: A Novel Mechanism for Red Recombination Keywords: Recombineering, Lambda Red, Recombination, Mechanism Corresponding Authors: Joshua A. Mosberg and Marc J. Lajoie Department of Genetics Harvard Medical School 77 Avenue Louis Pasteur New Research Building, Room 238 Boston, MA 02115 Phone: (617) 432-7670 Fax: (617) 432-6513 E-mails: [email protected], [email protected] 2 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 5′ ends of double-stranded DNA are recessed by Lambda Exonuclease, leaving behind 3′ 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 co-segregating 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. INTRODUCTION 3 Over the past decade, Lambda Red recombination (“recombineering”) has been used as a powerful technique for making precisely defined insertions, deletions, and point mutations in Escherichia coli, requiring as few as 35 base pairs of homology on each side of the desired alteration (SHARAN et al. 2009; THOMASON et al. 2007a). With this system, single-stranded DNA (ssDNA) oligonucleotides have been used to efficiently modify E. coli chromosomal targets (COSTANTINO and COURT 2003; ELLIS et al. 2001), BACs (SWAMINATHAN et al. 2001), and plasmids (THOMASON et al. 2007b), as well as to rapidly optimize a metabolic pathway coding for the production of lycopene (WANG et al. 2009). Furthermore, linear double-stranded DNA (dsDNA) recombineering has been used to replace chromosomal genes (MURPHY 1998; MURPHY et al. 2000), to disrupt gene function (DATSENKO and WANNER 2000), and to develop novel cloning methods (LEE et al. 2001; LI and ELLEDGE 2005). Large-scale dsDNA recombineering projects include creating a library of single-gene knockout E. coli strains (BABA et al. 2006) and removing 15% of the genomic material from a single E. coli strain (POSFAI et al. 2006). Linear dsDNA recombineering has also been used to insert heterologous genes and entire pathways into the E. coli chromosome (WANG and PFEIFER 2008; ZHANG et al. 1998) and BACs (LEE et al. 2001; WARMING et al. 2005), including those used for downstream applications in eukaryotes (BOUVIER and CHENG 2009; CHAVEROCHE et al. 2000). However, despite the broad use of this method, the mechanism of Lambda Red recombination has not achieved scientific consensus, particularly in the case of dsDNA recombination. A clearer understanding of the mechanism underlying this process could suggest ways to improve the functionality, ease, and versatility of Lambda Red recombination. Three phage-derived Lambda Red proteins are necessary for carrying out dsDNA recombination: Gam, Exo, and Beta. Gam prevents the degradation of linear dsDNA by the E. 4 coli RecBCD and SbcCD nucleases; Lambda Exonuclease (Exo) degrades dsDNA in a 5′ to 3′ manner, leaving single-stranded DNA in the recessed regions; and Beta binds to the single- stranded regions 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 5′ ends of the dsDNA and degrades in both directions simultaneously to produce a double-stranded region flanked on both sides by 3' 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). However, 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 recombination remains - highly proficient in a recA background (YU et al. 2000). Furthermore, a detailed analysis of Lambda Red recombination products showed characteristics 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 replication, which suggests strand annealing rather than strand invasion (LIM et al. 2008; POTEETE 2008). To explain these results, Court et al. (COURT et al. 2002) proposed a strand annealing model for insertional dsDNA recombination (Figure 1A), in which one single-stranded 3′ end anneals to its homologous target at the replication fork. The replication fork then stalls, due to the presence of a large dsDNA non-homology (i.e. the insertion cassette). The stalled replication fork is ultimately rescued by the other replication fork traveling in the opposite direction around the circular bacterial chromosome. The other 3′ end of the recombinogenic DNA anneals to the 5 homology region exposed by the second replication fork, forming a crossover structure, which is then resolved by unspecified E. coli enzymes (COURT et al. 2002). The Court mechanism was challenged by Poteete (POTEETE 2008), who showed that the dsDNA recombination of a linear Lambda phage chromosome occurs readily onto a unidirectionally-replicating plasmid, which does not have the second replication fork required by the Court mechanism (COURT et al. 2002). Thus, Poteete proposed an alternate mechanism (POTEETE 2008), termed “replisome invasion” (Figure 1B), in which a 3′ overhang of the Exo- processed dsDNA first anneals to its complementary sequence on the lagging strand of the recombination target. Subsequently, this overhang displaces the leading strand, thereby serving as the new template for leading strand synthesis. The resulting structure is resolved by an unspecified endonuclease, after which the recombinogenic DNA becomes the template for the synthesis of both new strands. In the context of recombineering using a linear dsDNA cassette, the author indicates that a second strand switching event must occur at the other end of the incoming dsDNA. While Poteete's mechanism addresses some of the weaknesses of the Court mechanism, it remains largely speculative. This mechanism does not identify the endonuclease responsible for resolving the structure after the first template switching event, nor does it explain how the recombinogenic DNA and replication machinery form a new replication fork. Additionally, this template switching mechanism would have to operate two times in a well-controlled manner, which may not be consistent with the high recombination frequencies often observed (MURPHY et al. 2000) for Lambda Red-mediated dsDNA insertion. Finally, little experimental evidence has been advanced to directly support this hypothesis. 6 To address the deficiencies in these mechanisms, we propose that Lambda Red dsDNA recombination proceeds via a ssDNA intermediate rather than a dsDNA core flanked by 3' overhangs (Figure 2). In this mechanism, Exo binds to one of the two dsDNA strands and degrades that strand completely, leaving behind full-length ssDNA. This ssDNA then anneals to its homology target at the lagging strand of the replication fork, and is incorporated as part of the newly-synthesized strand as if it were an Okazaki fragment. This process is analogous to the accepted mechanism for the Lambda Red-mediated recombination of ssDNA oligonucleotides (COURT et al. 2002), and therefore unifies the mechanisms for ssDNA and dsDNA recombination. Notably, our mechanism uses one replication fork for the incorporation of a full- length heterologous cassette, thereby addressing Poteete’s criticism of the Court mechanism. The degradation of an entire strand by Lambda Exo is feasible, given the highly processive nature of the enzyme (SUBRAMANIAN et al. 2003). Whereas previously proposed mechanisms assume that both dsDNA ends are degraded approximately simultaneously, our hypothesis implies that some dsDNA molecules will be