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EcoSal Plus
Domain 7:
Genetics and Genetic Tools
λ Recombination and Recombineering
MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.
- Author: Kenan C. Murphy1
- Editor: James M. Slauch2
-
VIEW AFFILIATIONS HIDE AFFILIATIONSAffiliations: 1: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01605; 2: The School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL
-
Received 13 August 2015 Accepted 04 November 2015 Published 11 January 2016
- Address correspondence to Kenan C. Murphy [email protected]
-
[open-access] The author has paid a fee to allow immediate free access to this article.
-
Abstract:
The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.
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Citation: Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
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Abstract:
The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.
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λ Recombination and Recombineering
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Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig1
Figure 1
The trimeric structure of λ Exo. View of the λ Exonuclease trimer looking through the central channel (A) and the same view rotated 90° to the right (B). The three subunits are colored blue, green, and magenta. The dsDNA passes through the central channel of the trimer, is acted upon by one of three active sites, and exits out the back as ssDNA. The structures were generated by PyMol based on the coordinates described by Zhang et al. (
53
).
ecosalplus/7/1/ESP-0011-2015_fig_001_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_001.gif
Click to view
Figure 1
The trimeric structure of λ Exo. View of the λ Exonuclease trimer looking through the central channel (A) and the same view rotated 90° to the right (B). The three subunits are colored blue, green, and magenta. The dsDNA passes through the central channel of the trimer, is acted upon by one of three active sites, and exits out the back as ssDNA. The structures were generated by PyMol based on the coordinates described by Zhang et al. ( 53 ).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig2
Figure 2
Models for λ Beta-DNA structures. (A) A large Beta ring (18 subunits) is shown with DNA wrapped around the outside of the ring, as previously suggested for P22 Erf (
64
). (B) After Beta-catalyzed annealing of complementary ssDNA strands, Beta-dsDNA filaments are formed. The authors estimate the Beta filament contains around 100 base pairs per supercoil turn of the DNA. Taken from Passy et al. (
68
), with permission.
ecosalplus/7/1/ESP-0011-2015_fig_002_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_002.gif
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Figure 2
Models for λ Beta-DNA structures. (A) A large Beta ring (18 subunits) is shown with DNA wrapped around the outside of the ring, as previously suggested for P22 Erf ( 64 ). (B) After Beta-catalyzed annealing of complementary ssDNA strands, Beta-dsDNA filaments are formed. The authors estimate the Beta filament contains around 100 base pairs per supercoil turn of the DNA. Taken from Passy et al. ( 68 ), with permission.
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig3
Figure 3
Ribbon diagram of the λ Gam protein dimer; chains A and B labeled green and magenta. It is an all α-helical protein with a dimerization domain (center region) and two protruding N-terminal helices (H1), sticking out at an angle of about 100° from each other. A proposed conformational change occurs upon binding of λ Gam to RecBCD, with the H1 helices rotating about 120° around the Gly-Ile-Pro hinge regions (denoted by arrow in the green subunit). The proposed conformation change places the H1 helices of each subunit into the ssDNA binding regions of RecB and RecCD, thus inhibiting binding of RecBCD to dsDNA ends. Structure generated by PyMol based on the coordinates described by Court et al. (
83
).
ecosalplus/7/1/ESP-0011-2015_fig_003_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_003.gif
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Figure 3
Ribbon diagram of the λ Gam protein dimer; chains A and B labeled green and magenta. It is an all α-helical protein with a dimerization domain (center region) and two protruding N-terminal helices (H1), sticking out at an angle of about 100° from each other. A proposed conformational change occurs upon binding of λ Gam to RecBCD, with the H1 helices rotating about 120° around the Gly-Ile-Pro hinge regions (denoted by arrow in the green subunit). The proposed conformation change places the H1 helices of each subunit into the ssDNA binding regions of RecB and RecCD, thus inhibiting binding of RecBCD to dsDNA ends. Structure generated by PyMol based on the coordinates described by Court et al. ( 83 ).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig4
Figure 4
Two classic pathways of λ Red-promoted phage recombination. dsDNA ends of the phage chromosome are provided by the action of terminase. λ Exo (red trapezoid) binds to a dsDNA end and digests the 5′ strand, assisting Beta (blue ring made of small circles) to bind to the 3′-ssDNA tail. (A) The RecA-dependent pathway: In the absence of replication, Beta is replaced with RecA (yellow triangle) with the help of RecF pathway functions, which promotes invasion of the ssDNA into a homologous duplex. Recombination proceeds via branch migration, Holliday junction formation, and subsequent resolution of the intermediate by the host resolvasome, RuvABC. (B) The ssDNA annealing pathway: dsDNA ends are formed containing terminal redundancies, generated by the rolling-circle mode of replication and/or terminase cutting during the lytic infection. Exo and Beta process the ends as above. The Beta protein promotes annealing between the overlapping ssDNA ends, which are filled in by DNA polymerase I and ligated together to form a recombinant.
ecosalplus/7/1/ESP-0011-2015_fig_004_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_004.gif
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Figure 4
Two classic pathways of λ Red-promoted phage recombination. dsDNA ends of the phage chromosome are provided by the action of terminase. λ Exo (red trapezoid) binds to a dsDNA end and digests the 5′ strand, assisting Beta (blue ring made of small circles) to bind to the 3′-ssDNA tail. (A) The RecA-dependent pathway: In the absence of replication, Beta is replaced with RecA (yellow triangle) with the help of RecF pathway functions, which promotes invasion of the ssDNA into a homologous duplex. Recombination proceeds via branch migration, Holliday junction formation, and subsequent resolution of the intermediate by the host resolvasome, RuvABC. (B) The ssDNA annealing pathway: dsDNA ends are formed containing terminal redundancies, generated by the rolling-circle mode of replication and/or terminase cutting during the lytic infection. Exo and Beta process the ends as above. The Beta protein promotes annealing between the overlapping ssDNA ends, which are filled in by DNA polymerase I and ligated together to form a recombinant.
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig5
Figure 5
Substrates used to demonstrate Red-promoted replisome invasion. Recombination occurs between a replicating resident target plasmid (direction of replication shown by black triangle) and a nonreplicating λ chromosome. The homologous regions are denoted by the green box. The λ chromosome is delivered at high efficiency by infection, is inhibited from replicating by overexpression of the λ c1 repressor, and is cut in vivo by a chromosomally encoded PaeR7 restriction enzyme. DNA from the infected cells is isolated at different times after infection, cut with BamHI, and subjected to a Southern procedure. The amount of recombinant band (bottom) is detected by probing a Southern blot for sequences designated “P.” (Descriptions of substrates were derived from reference
3
).
ecosalplus/7/1/ESP-0011-2015_fig_005_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_005.gif
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Figure 5
Substrates used to demonstrate Red-promoted replisome invasion. Recombination occurs between a replicating resident target plasmid (direction of replication shown by black triangle) and a nonreplicating λ chromosome. The homologous regions are denoted by the green box. The λ chromosome is delivered at high efficiency by infection, is inhibited from replicating by overexpression of the λ c1 repressor, and is cut in vivo by a chromosomally encoded PaeR7 restriction enzyme. DNA from the infected cells is isolated at different times after infection, cut with BamHI, and subjected to a Southern procedure. The amount of recombinant band (bottom) is detected by probing a Southern blot for sequences designated “P.” (Descriptions of substrates were derived from reference 3 ).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig6
Figure 6
Replisome invasion/template switch model of λ recombination. The diagram depicts a recombination event between the tip of a rolling circle (circle not shown), and another replicon (either one of the replisomes of a theta-mode intermediate, or the replisome of a second rolling circle). (Top) A Red-processed dsDNA end (Beta bound to a ssDNA overhang generated by Exo) invades a replication fork and promotes annealing to the lagging strand template. (Middle) Beta captures the leading strand and promotes a template switch, such that the leading strand polymerase now uses the incoming strand as a template. (Bottom) Template switch (TS) model invokes a redirection of the replisome to the incoming strand. The template switch then connects one arm of the original replisome to the invading duplex (i.e., the recombination event). As before, red trapezoid, λ Exo; blue circles, λ Beta. Yellow oval represents the replisome.
ecosalplus/7/1/ESP-0011-2015_fig_006_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_006.gif
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Figure 6
Replisome invasion/template switch model of λ recombination. The diagram depicts a recombination event between the tip of a rolling circle (circle not shown), and another replicon (either one of the replisomes of a theta-mode intermediate, or the replisome of a second rolling circle). (Top) A Red-processed dsDNA end (Beta bound to a ssDNA overhang generated by Exo) invades a replication fork and promotes annealing to the lagging strand template. (Middle) Beta captures the leading strand and promotes a template switch, such that the leading strand polymerase now uses the incoming strand as a template. (Bottom) Template switch (TS) model invokes a redirection of the replisome to the incoming strand. The template switch then connects one arm of the original replisome to the invading duplex (i.e., the recombination event). As before, red trapezoid, λ Exo; blue circles, λ Beta. Yellow oval represents the replisome.
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig7
Figure 7
New replication fork model of λ recombination. As in
Fig. 6
, the annealing of the ssDNA generated by λ Exo anneals to an ssDNA region on the lagging strand template. In this model, however, the invaded replisome is not affected. Instead, the invasion of the incoming duplex initiates a new fork that travels in the opposite direction, with the annealed strand becoming the template for the new fork’s lagging strand. The incoming duplex is then connected to one arm of the fork (i.e., the recombination event). As before, red trapezoid, λ Exo; blue circles, λ Beta. Yellow oval represents the replisome.
ecosalplus/7/1/ESP-0011-2015_fig_007_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_007.gif
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Figure 7
New replication fork model of λ recombination. As in Fig. 6 , the annealing of the ssDNA generated by λ Exo anneals to an ssDNA region on the lagging strand template. In this model, however, the invaded replisome is not affected. Instead, the invasion of the incoming duplex initiates a new fork that travels in the opposite direction, with the annealed strand becoming the template for the new fork’s lagging strand. The incoming duplex is then connected to one arm of the fork (i.e., the recombination event). As before, red trapezoid, λ Exo; blue circles, λ Beta. Yellow oval represents the replisome.
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig8
Figure 8
Diagram showing how the 3′-ssDNA tails of dsDNA substrates (generated by λ Exo acting on the dsDNA ends) could anneal to the ssDNA regions of a replication fork. The 3′-ssDNA tail on top (in green) anneals to an ssDNA region within the lagging strand template, while the 3′-ssDNA tail on bottom (in red) anneals to an ssDNA region within the leading strand template (a more infrequent event perhaps, due to lesser amounts of ssDNA expected on this template). In either case, the invading duplex becomes one prong of the new fork, with the annealed strand becoming the new lagging strand template.
ecosalplus/7/1/ESP-0011-2015_fig_008_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_008.gif
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Figure 8
Diagram showing how the 3′-ssDNA tails of dsDNA substrates (generated by λ Exo acting on the dsDNA ends) could anneal to the ssDNA regions of a replication fork. The 3′-ssDNA tail on top (in green) anneals to an ssDNA region within the lagging strand template, while the 3′-ssDNA tail on bottom (in red) anneals to an ssDNA region within the leading strand template (a more infrequent event perhaps, due to lesser amounts of ssDNA expected on this template). In either case, the invading duplex becomes one prong of the new fork, with the annealed strand becoming the new lagging strand template.
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig9
Figure 9
Gene replacement and verification of recombinants using recombineering. (A) Outline of the basic steps involved in recombineering. (B) Primer design for gene replacement and verification. The 3′ ends of primers 1 and 2 contain 20 bp for amplification of the drugR marker (including regulatory regions), while the 5′ ends of the primers contain 40 to 50 bp of sequence that flank the target gene (red lines). Primers 5 and 6 are used to verify the 5′ junction of the recombinant, and primers 7 and 8 are used to verify the 3′ junction of the recombinant. Primers 5 and 8 can be used to verify loss of wild-type sequence (either by agarose gel or restriction enzyme analysis). Alternatively, primers 3 and 4 are designed to generate an internal fragment of the target gene. This product should be absent in the recombinant.
ecosalplus/7/1/ESP-0011-2015_fig_009_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_009.gif
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Figure 9
Gene replacement and verification of recombinants using recombineering. (A) Outline of the basic steps involved in recombineering. (B) Primer design for gene replacement and verification. The 3′ ends of primers 1 and 2 contain 20 bp for amplification of the drugR marker (including regulatory regions), while the 5′ ends of the primers contain 40 to 50 bp of sequence that flank the target gene (red lines). Primers 5 and 6 are used to verify the 5′ junction of the recombinant, and primers 7 and 8 are used to verify the 3′ junction of the recombinant. Primers 5 and 8 can be used to verify loss of wild-type sequence (either by agarose gel or restriction enzyme analysis). Alternatively, primers 3 and 4 are designed to generate an internal fragment of the target gene. This product should be absent in the recombinant.
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig10
Figure 10
The use of sacB as a counterselection marker in recombineering. The cat-sacB cassette is used as a template for PCR to generate an amplicon that has the cassette flanked by 50 bp of target homology. The recombineering event can either insert the cassette into the target gene, or replace sequences within the target gene with the cassette. After selection for chloramphenicol resistance and verification of sucrose sensitivity, the modified strain is electroporated with either a dsDNA substrate or an oligo that contains the desired mutation (red rectangle). The modified strain is selected by resistance to sucrose and screened for sensitivity to chloramphenicol.
ecosalplus/7/1/ESP-0011-2015_fig_0010_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_0010.gif
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Figure 10
The use of sacB as a counterselection marker in recombineering. The cat-sacB cassette is used as a template for PCR to generate an amplicon that has the cassette flanked by 50 bp of target homology. The recombineering event can either insert the cassette into the target gene, or replace sequences within the target gene with the cassette. After selection for chloramphenicol resistance and verification of sucrose sensitivity, the modified strain is electroporated with either a dsDNA substrate or an oligo that contains the desired mutation (red rectangle). The modified strain is selected by resistance to sucrose and screened for sensitivity to chloramphenicol.
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig11
Figure 11
Gene replacement and Cre-mediated marker eviction (see text for details).
ecosalplus/7/1/ESP-0011-2015_fig_0011_thmb.gif
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Figure 11
Gene replacement and Cre-mediated marker eviction (see text for details).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig12
Figure 12
Cre-mediated large deletion (see text for details).
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Figure 12
Cre-mediated large deletion (see text for details).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig13
Figure 13
I-SceI-induced deletion (see text for details).
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Figure 13
I-SceI-induced deletion (see text for details).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig14
Figure 14
Insertion of foreign DNA into the E. coli chromosome (see text for details).
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Figure 14
Insertion of foreign DNA into the E. coli chromosome (see text for details).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig15
Figure 15
Red-mediated duplication with establishment of new forks (see text for details).
ecosalplus/7/1/ESP-0011-2015_fig_0015_thmb.gif
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Figure 15
Red-mediated duplication with establishment of new forks (see text for details).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig16
Figure 16
Red-mediated duplication with fork disruption (see text for details).
ecosalplus/7/1/ESP-0011-2015_fig_0016_thmb.gif
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Figure 16
Red-mediated duplication with fork disruption (see text for details).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig17
Figure 17
Chromosomal mutagenesis with λ Red (see text for details).
ecosalplus/7/1/ESP-0011-2015_fig_0017_thmb.gif
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Figure 17
Chromosomal mutagenesis with λ Red (see text for details).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig18
Figure 18
Regulatory region engineering (see text for details).
ecosalplus/7/1/ESP-0011-2015_fig_0018_thmb.gif
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Figure 18
Regulatory region engineering (see text for details).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig19
Figure 19
λ Red Recombineering of non-E. coli sequences. Exogenous DNAs (e.g., mycobacteria, mouse, or human DNA) are cloned into bacterial artificial chromosomes (BACs) for modifications, additions, or deletions. Schemes to recover the modified DNA for sending it back into the exogenous hosts are discussed in the text. ES cells, embryonic stem cells.
ecosalplus/7/1/ESP-0011-2015_fig_0019_thmb.gif
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Figure 19
λ Red Recombineering of non-E. coli sequences. Exogenous DNAs (e.g., mycobacteria, mouse, or human DNA) are cloned into bacterial artificial chromosomes (BACs) for modifications, additions, or deletions. Schemes to recover the modified DNA for sending it back into the exogenous hosts are discussed in the text. ES cells, embryonic stem cells.
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig20
Figure 20
Beta-promoted Recombineering with ssDNA oligos. (A–C) Targeting of the lagging strand template. Beta promotes annealing of the oligo to a ssDNA region of the lagging strand template. Pol I and ligase promote filling in of the oligo and joining it with the surrounding Okazaki fragments to produce heteroduplex DNA (red asterisk). (D–F) Targeting of the leading strand template. Beta promotes annealing of the oligo to a ssDNA region of the leading strand template just ahead of the leading strand 3′ end. The leading strand polymerase (not shown) dissociates from its template, and because it is tethered to the clamp loader (green pentagon) of the replisome (denoted by yellow oval), it can reinitiate downstream at the 3′ end of the oligo. Annealing of the oligo creates either a mismatch, a small deletion, or small insertion (denoted by red asterisk), which must escape repair by the Mismatch Repair System of E. coli. The mutation is fixed via subsequent replication of the heteroduplex region.
ecosalplus/7/1/ESP-0011-2015_fig_0020_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_0020.gif
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Figure 20
Beta-promoted Recombineering with ssDNA oligos. (A–C) Targeting of the lagging strand template. Beta promotes annealing of the oligo to a ssDNA region of the lagging strand template. Pol I and ligase promote filling in of the oligo and joining it with the surrounding Okazaki fragments to produce heteroduplex DNA (red asterisk). (D–F) Targeting of the leading strand template. Beta promotes annealing of the oligo to a ssDNA region of the leading strand template just ahead of the leading strand 3′ end. The leading strand polymerase (not shown) dissociates from its template, and because it is tethered to the clamp loader (green pentagon) of the replisome (denoted by yellow oval), it can reinitiate downstream at the 3′ end of the oligo. Annealing of the oligo creates either a mismatch, a small deletion, or small insertion (denoted by red asterisk), which must escape repair by the Mismatch Repair System of E. coli. The mutation is fixed via subsequent replication of the heteroduplex region.
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig21
Figure 21
Red-promoted dsDNA recombineering via ssDNA intermediates. λ Exo binds to one end of a dsDNA substrate and degrades the 5′-ending strand. The long ssDNA generated by Exo is bound by Beta (exactly how and to what extent is not known). Beta then promotes annealing to ssDNA regions of the replication fork, much like the model in
Fig. 20
for DNA oligos. The large nonhomologous ssDNA region encoding the drugR marker (brown line) is presumably stabilized by Beta bound to regions flanking the nonhomology. When the next replication fork passes through, the gene replacement is completed. This model was originally proposed by Yu et al. (
376
), and corroborated by the studies of Mosberg et al. (
378
).
ecosalplus/7/1/ESP-0011-2015_fig_0021_thmb.gif
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Figure 21
Red-promoted dsDNA recombineering via ssDNA intermediates. λ Exo binds to one end of a dsDNA substrate and degrades the 5′-ending strand. The long ssDNA generated by Exo is bound by Beta (exactly how and to what extent is not known). Beta then promotes annealing to ssDNA regions of the replication fork, much like the model in Fig. 20 for DNA oligos. The large nonhomologous ssDNA region encoding the drugR marker (brown line) is presumably stabilized by Beta bound to regions flanking the nonhomology. When the next replication fork passes through, the gene replacement is completed. This model was originally proposed by Yu et al. ( 376 ), and corroborated by the studies of Mosberg et al. ( 378 ).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig22
Figure 22
Proposed model for integration of dsDNA substrate with limited nonhomology into a replication fork. (A) Red-processed dsDNA end (Beta bound to a ssDNA overhang generated by Exo) invades a replication fork and promotes annealing to the lagging strand template. (B) The leading strand switches to the incoming duplex. Stalling and reversal of the fork generates a “chicken-foot” structure. (C) The leading strand polymerase fills in the gap previously generated by λ Exo, while DNA ligase connects the strands. (D) The Holliday junction branch migrates to the right and is reabsorbed, reestablishing a replication fork framework. The mutant base(s) form heteroduplexes (green boxes). Model taken from Court et al. (
381
).
ecosalplus/7/1/ESP-0011-2015_fig_0022_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_0022.gif
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Figure 22
Proposed model for integration of dsDNA substrate with limited nonhomology into a replication fork. (A) Red-processed dsDNA end (Beta bound to a ssDNA overhang generated by Exo) invades a replication fork and promotes annealing to the lagging strand template. (B) The leading strand switches to the incoming duplex. Stalling and reversal of the fork generates a “chicken-foot” structure. (C) The leading strand polymerase fills in the gap previously generated by λ Exo, while DNA ligase connects the strands. (D) The Holliday junction branch migrates to the right and is reabsorbed, reestablishing a replication fork framework. The mutant base(s) form heteroduplexes (green boxes). Model taken from Court et al. ( 381 ).
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.fig23
Figure 23
Template switch model of recombineering. (A) As before, a Red-processed dsDNA end (Beta bound to a ssDNA overhang generated by Exo) invades a replication fork and promotes annealing to the lagging strand template. (B) Beta captures the leading strand and promotes a template switch, such that the leading strand polymerase now uses the incoming strand as a template. A nick is introduced in the leading strand template by an unspecified nuclease. (C) The redirected polymerase completely resynthesizes the incoming strand, reestablishing a dsDNA end. The 3′ end of the invading strand is filled in and ligated to complete the recombination event. (D) The products of the reaction are an intact chromosome (after filling in and ligation) and the broken end containing the incoming substrate. This dsDNA end could be acted upon by the λ Red system and invade the leading strand template of another replisome (for small homology substrates), or by the RecA-dependent pathway for recombination (for long homology substrates) to complete the gene replacement.
ecosalplus/7/1/ESP-0011-2015_fig_0023_thmb.gif
ecosalplus/7/1/ESP-0011-2015_fig_0023.gif
Click to view
Figure 23
Template switch model of recombineering. (A) As before, a Red-processed dsDNA end (Beta bound to a ssDNA overhang generated by Exo) invades a replication fork and promotes annealing to the lagging strand template. (B) Beta captures the leading strand and promotes a template switch, such that the leading strand polymerase now uses the incoming strand as a template. A nick is introduced in the leading strand template by an unspecified nuclease. (C) The redirected polymerase completely resynthesizes the incoming strand, reestablishing a dsDNA end. The 3′ end of the invading strand is filled in and ligated to complete the recombination event. (D) The products of the reaction are an intact chromosome (after filling in and ligation) and the broken end containing the incoming substrate. This dsDNA end could be acted upon by the λ Red system and invade the leading strand template of another replisome (for small homology substrates), or by the RecA-dependent pathway for recombination (for long homology substrates) to complete the gene replacement.
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
Tables
λ Recombination and Recombineering
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Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.T1
Table 1
Plasmids expressing λ red and gam
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Table 1
Plasmids expressing λ red and gam
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.T2
Table 2
Drug-resistance cassettes used for λ Red recombineering
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Table 2
Drug-resistance cassettes used for λ Red recombineering
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
/content/ecosalplus/10.1128/ecosalplus.ESP-0011-2015.T3
Table 3
Cre- and Flp-expressing plasmids
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Table 3
Cre- and Flp-expressing plasmids
Citation:
Murphy K. 2016. λ Recombination and Recombineering, EcoSal Plus 2016; doi:10.1128/ecosalplus.ESP-0011-2015
Supplemental Material
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