ET - Recombination. Introduction

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1 GENERAL & APPLIED GENETICS Geert Van Haute August 2003 ET - Recombination Introduction Homologous recombination is of importance to a variety of cellular processes, including the maintenance of genomic integrity, proper segregation of chromosomes in meiosis, and the rescue of stalled replication forks. Homologous recombination provides a mean for repair of DNA double-stranded breaks (DSBs), which can arise during DNA replication as well as after damage by external factors such as irradiation. If DSBs are not repaired, derangements including severe chromosomal defects, carcinogenesis, and cell death can occur. To date studies of DSB repair by homologous recombination have focused on pathways initiated by the prokaryotic protein RecA or its eukaryotic homolog RAD51. However, alternative homologous recombination pathways, which function independently of RecA or RAD51, have been described. For example, inactivation of the RecA/RecBCD pathway in Escherichia coli recbc mutants can be suppressed by sbca or sbcbc mutations. In sbca strains, a cryptic Rac prophage operon is activated to express RecE and RecT. RecE is a 5 3 exonuclease and RecT is a ssdna-binding protein that promotes ssdna annealing, strand transfer, and strand invasion in vitro. λ Phage contains a similar system to mediate homologous recombination independently of RecA. Here also a phage operon encodes a 5 3 exonuclease and a ssdna-binding protein with annealing and strand exchange activity. A variety of studies have concluded that RecE/RecT and Redα/Redβ are functionally equivalent. In particular, rece/rect can substitute for the Redα/Redβ genes in λ recombination. Figure 1 : function of RecA 1

2 Principle Two models that are not exclusive of each other have been developed to explain DSB repair initiated by the RecE/RecT and Redα/Redβ pathways. In both models, the 5 3 exonuclease, RecE or Redα, resects a DSB to expose a 3 -ended single-stranded region that is then bound by the annealing protein, RecT or Redβ. Fig. 2 Current model for double-stranded-break repair initiated by RecE/RecT and Redα/Redβ. For clarity, only one linear end is shown. First, RecE or Redα degrades the DNA in a 5 3 direction, starting from the DSB, thereby creating a 3 ssdna overhang. Then, RecT or Redβ binds to the ssdna, forming a recombinogenic proteonucleic filament which is used in recombination, either by single strand annealing or by strand invasion. 1. In the annealing model, the protein ssdna filament anneals to a complementary single-stranded region that has arisen from either a similarly prepared DSB or from a DSB produced by DNA replica 2. In the strand invasion model, the protein ssdna filament establishes a D-loop in an unbroken DNA region. The initial steps of double-stranded break (DSB) repair by homologous recombination mediated by the 5 3 exonuclease/annealing protein pairs, RecE/RecT and Redα/Redβ, were analyzed. Recombination was RecA-independent and required the expression of both components of an orthologous pair, even when the need for exonuclease activity was removed by use of preresected substrates. The required orthologous function correlated with a specific protein protein interaction, and recombination was favored by overexpression of the annealing protein with respect to the exonuclease. The need for both components of an orthologous pair was observed regardless of whether recombination proceeded via a singlestrand annealing or a putative strand invasion mechanism. The DSB repair reactions studied here are reminiscent of the RecBCD/RecA reaction and suggest a general mechanism that is likely to be relevant to other systems, including RAD52 mediated recombination. 2

3 Homologous recombination is of importance to a variety of cellular processes, including the maintenance of genomic integrity, proper segregation of chromosomes in meiosis, and the rescue of stalled replication forks. Candidate E. coli hosts were coelectroporated with an intact circular plasmid and a PCR product synthesized to include short flanking regions of homology to the plasmid. Only sbca hosts gave workable rates of homologous recombination. Plasmids expressing the rece/rect or redα/redβ genes convey efficient homologous recombination to recbc hosts (1,2). The use of these protein pairs in a linear plus circular DSB repair reaction is refered to as ET recombination. Both components of either the RecE/RecT or the Redα/Redβ system are required. Figure 3: ET recombination assay To examine recombination initiation by RecE/RecT and Redα/Redβ, the ET recombination assay illustrated in Figure 3 was used. The gene(s) for the protein(s) of interest, the genes RecE, RecT, Redα, Redβ, were cloned behind the L-arabinose inducible BAD promoter of pbad24. After L-arabinose induction, electrocompetent cells carrying these constructs were prepared and transformed with PCR-generated linear molecules carrying the chloramphenicol resistance gene cmr flanked by homology regions of 50 nucleotides (denoted a and b). They were introduced into host strains containing plasmids from which different combinations of RecE, RecT, Redα and/or Redβ were expressed. These homology regions were also present on the circular target, directly flanking the ampicillin resistance gene bla. The homology regions directed recombination into the expression plasmid itself. Substitution of bla by cmr via recombination through the homology regions resulted in the expression of cmr from the bla promoter, giving rise to chloramphenicol resistant colonies. Virtually all colonies that 3

4 acquired antibiotic resistance carried the intended homologously recombined product as determined by DNA restriction analysis (1,6). RecA cannot substitute for RecT or Redβ Recombination proceeds efficiently only when both RecE and RecT, or Redα and Redβ, are coexpressed. To test whether RecA contributed to recombination in this assay, or could substitute for any component, parallel experiments were performed in a reca + host, JC5519. Very similar results were obtained in the presence and absence of RecA. In this assay, RecA cannot substitute for RecT or Redα, nor affect recombination efficiency. (see fig. 4a, 4b) Figure 4a: Recombination in JC5547 (reca -, recbc - ) mediated by RecE (E), RecT (T), RecE/RecT (ET), Red α (α), Redβ (β), Redα/Redβ (βα), or no exogenous protein(1) Figure 4b : Recombination in JC5519 (reca +, recbc - ).(1) The cloning of foreign DNA in Escherichia coli episomes (plasmids, BACs, etc.) is a cornerstone of molecular biology. Using DNA ligases to paste DNA into vectors, is still the most widely used approach. The place where fragments can be inserted is limited by the presence of restriction sites. The length of fragments that can be ligated is also limited. ET recombination, first described in 1998 (1), is a technique for directed cloning and subcloning that permits a chosen DNA region to be cloned from a complex mixture without prior isolation. There for cloning by ET recombination resembles PCR in that both involve the amplification of a DNA region between two chosen points. The approach is termed "ET recombination" because it was first uncovered, using the Rec phage protein pair, RecE/RecT. Later it was shown that the equivalent lambda phage protein pair, Redα /Redβ, also worked (1,2). The researchers original goal was to develop a simple method to engineer bacterial artificial chromosomes (BACs). The approach worked so well that other applications became apparent, such as rapid creation of new E. coli strains through direct targeting of the E. coli chromosome (1), rapid generation of gene targeting constructs for use in mouse embryonic stem (ES) cells (5), and a new way to accomplish site-directed mutagenesis in plasmids (1) and BACs (4). To date, all applications of ET recombination, whether with RecE/RecT or Redα /Redβ have modified pre-existing replication-competent molecules. Here we show that ET recombination can be applied to clone and subclone DNA regions from a DNA source into a plasmid. This new application provokes very different practical implications. 4

5 ET recombination works well with homology regions that are short enough to be included in synthetic oligonucleotides (1).These oligonucleotides can also contain a primer site for PCR amplification of a selectable gene, such as an antibiotic resistance gene. Hence the PCR product contains the selectable gene flanked by two homology arms. In the presence of either RecE/RecT or Redα /Redβ and the absence of RecBCD, homologous recombination between the homology arms and the chosen target regions integrates the selectable gene. In other words, the homology arms define the integration site, and thereby an existing replicationcompetent molecule is modified (1). This strategy can be altered so that the PCR product is a plasmid backbone, including origin of replication and selectable gene, flanked by homology arms(3). In this application, the homology arms define the region that is to be copied into the plasmid. To test whether ET recombination could be used in this way, the researchers first tried subcloning various regions from replication-competent molecules present in E. coli. The experimental strategy is illustrated in Figure 1. Figure 5. Subcloning by ET recombination Figure 5 is showing the linear cloning vector carrying an E. coli plasmid origin and an antibiotic selectable marker (Sm) gene flanked by two oligonucleotide homology arms. The linear cloning vectors were PCR amplified using oligonucleotides containing the homology arms at their 5 end, and PCR primers at their 3 ends for amplification of the plasmid origin/antibiotic resistance gene cassettes. Several variations and targets were tested by choosing different homology arms (by oligonucleotide synthesis) to flank various target regions, including the endogenous lacz gene on the E. coli chromosome, a part of a highcopy plasmid, and parts of a BAC. The PCR products included the p15a plasmid origin combined with different selectable genes. They were electroporated into ET-competent E. coli hosts that carried the respective targets. Antibiotic-resistant colonies were examined for the intended recombination event, which was very efficient. In all cases, the complete region between the homology arms was fully inserted into the episome without any detectable mutational errors.(3) Empty circularisation products were observed and they accounted for all of the incorrect resistant colonies examined. Empty circularisation is the most important source of background but, the intended ET recombinants were the most abundant products. (a range between 100% and 83%, each on 18 colonies, depending on the treatment) After these subcloning experiments, which showed that ET recombination is an efficient way to amplify a chosen target region from the complex back-ground presented by the E. coli genome, the researchers next applied ET recombination to the more difficult task of direct cloning from complex mixtures of exogenous DNA. 5

6 Figure 6. Cloning by ET recombination. Conclusion The researchers did not find any limitation of target choice according to the size or site. Any recipient in E. coli from high copy plasmid to the E. coli chromosome, appears ameble to precise alteration. In addition to engineering large DNA, other restriction endonucleaseindependent types of DNA alteration become feasible. For example, deletions between any chosen base pairs in a target episome can be made by choice of oligonucleotide homology arms. Similarly, chosen DNA sequences can be inserted at a chosen base pair to create, for example, altered protein reading frames. Combinations of insertions and deletions, as well as point mutations are also possible. references 1. Zhang, Y., Buchholz, F., Muyrers, J.P.P. & Stewart, A.F. (1998). A new logic for DNA engineering using recombination in Escherichia coli. Nature Genetics Vol 20, p Muyrers J.P.P., Zhang, Y., Testa, G. & Stewart A.F. (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Research Vol 27, p Zhang Y., Muyrers J.P.P., Testa G., and Stewart A.F. (2000) DNA cloning by homologous recombination in Escherichia coli Nature Biotechnology Vol 18 p Muyrers, J.P.P, Zhang Y., Benes V., Testa G., Ansorge W. and Stewart A.F. (2000) Point mutation of bacterial artificial chromosomes by ET recombination EMBO reports Vol 1 no. 3 p Angrand, P.-O., Daigle, N., van der Hoeven, F., Scholer, H.R. & Stewart A.F. (1999). Simplified generation of targeting constructs using ET recombination. Nucleic Acids Research. Vol 27, p16 6. Muyrers J.P.P., Zhang Y., Buchholz F. and Stewart A.F. (2000) RecE/RecT and Redα/Redβ initiate double-stranded break repair by specifically interacting with their respective partners GENES & DEVELOPMENT vol.14 p Muyrers J.P.P., Zhang Y. and Stewart A.F. (2001) Techniques: Recombinogenic engineering new options for cloning and manipulating DNA TRENDS in Biochemical Sciences vol.26 No. 5 p