Difference between revisions of "DNA/Recombination"

Revision as of 01:13, 17 November 2008

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Site-specific DNA recombination requires both a recombinase protein and a pair of repeated DNA sites at which recombination takes place. If the DNA sites are oriented as direct repeats, then recombination generally results in deletion of the intervening DNA. If the DNA sites are instead oriented as inverted repeats, then recombination generally results in inversion of the intervening DNA. While some DNA recombination systems, such as Cre/lox only require the recombinase and the two DNA sites for recombination to occur, others either require or are modulated by additional accessory factors.

Site-specific recombination systems derived from Salmonella, different bacteriophages, and E. coli are all available from the Registry. For more details, see below.

Salmonella typhimurium-derived Hin/hix DNA recombination system

Karmella Haynes and the [http://parts.mit.edu/wiki/index.php/Davidson_2006 2006 Davidson College/Missouri Western iGEM team], designed and constructed a set of parts from the Salmonella typhimurium-derived DNA recombination system. You can read more about the 2006 Davidson/Missouri Western project in their open-access paper [http://www.jbioleng.org/content/2/1/8 Engineering bacteria to solve the Burnt Pancake Problem] published in the Journal of Biological Engineering Haynes. The following is excerpted from their paper.

In Salmonella, Hin DNA recombinase (BBa_J31000, BBa_J31001) catalyzes an inversion reaction that regulates the expression of alternative flagellin genes by switching the orientation of a promoter located on a 1 kb invertible DNA segment Zieg,Zieg80. Two palindromic 26 bp hix sequences flank the invertible DNA segment and serve as the recognition sites for cleavage and strand exchange. A ~70 bp cis-acting recombinational enhancer (RE) increases efficiency of protein-DNA complex formation (BBa_J3101) Johnson. We have reconstituted the genetic elements required for DNA inversion as a collection of modular genetic elements for use in E. coli. Our system is a proof-of-concept genetic computing device that manipulates plasmid DNA processors within living cells.

In Salmonella, the asymmetrical palindromic sequences hixL and hixR flank the invertible DNA segment and serve as the recognition sites for cleavage and strand exchange. Our system uses hixC (BBa_J44000), a composite symmetrical hix site that shows higher binding affinity for Hin and a 16-fold slower inversion rate than wild type sites hixL and hixR Lim,Moskowitz.

We have demonstrated that a modified Hin/hix DNA recombination system can be used in vivo to manipulate at least two adjacent hixC-flanked DNA segments; HinLVA and hixC are sufficient for DNA inversion activity. The Hin/hix DNA recombination system could be used for other biological engineering applications. We have developed a set of modular genetic elements, hixC (BBa_J44000), RE (BBa_J3101), and HinLVA (BBa_J31001), that expands the repertoire of molecular tools for enzyme-mediated DNA manipulation in vivo.

References

<biblio>

1. Zieg pmid=322276
2. Zieg80 pmid=6933466
3. Johnson pmid=2548848
4. Haykinson pmid=8508775
5. PerkinsBalding pmid=9244261
6. Haynes pmid=18492232
7. Ham pmid=18665232
8. Nanassy pmid=9691026
9. Moswitz pmid=1885005
10. Lim pmid=1597453

</biblio>

Bacteriophage P1 Cre/lox DNA recombination system

Chris Anderson, a professor of bioengineering at UC Berkeley, constructed the lox recombination site BBa_J61046.

Eimad Shotar, a member of the [http://parts.mit.edu/igem07/index.php/Paris 2007 Paris iGEM team], constructed the lox recombination sites BBa_I718016 and BBa_I718017.

The following text is excerpted from Siegel et al. Siegel04.

Bacteriophage P1 uses a site-specific recombination system that is responsible for partitioning newly synthesized genomic copies during replication Abremski, Hoess. This system is composed of a 38-kD phage-encoded Cre recombinase that mediates symmetrical recombination between two 34-bp loxP sites Abremski, which are recreated after recombination. Recombination between two compatible loxP sites will excise or invert the intervening DNA in the case of an intramolecular reaction or transfer suitably flanked loxP DNA in an intermolecular double cross-over recombination event. The Cre/loxP system does not require accessory factors to carry out recombination in vivo or in vitro, and studies have identified several hetero-specific loxP sequences that exclusively recombine with themselves, but not with wild-type lox Hoess86, Sauer92, Lee98, Siegel01. Importantly, the Cre/lox system has also been shown to be functional in site-specific recombination in mammalian cell lines Sauer88.

References

<biblio>

1. Abremski pmid=6319400
2. Hoess pmid=6230671
3. Hamilton pmid=6333513
4. Hoess86 pmid=3457367
5. Sauer88 pmid=2839833
6. Sauer92 pmid=1554399
7. Lee98 pmid=9714735
8. Sauer98 pmid=9608509
9. Siegel01 pmid=11576551
10. Sauer02 pmid=12624421
11. Siegel04 pmid=15173117

</biblio>

Bacteriophage λ att DNA recombination system

The 2004 Boston University iGEM team designed and constructed the att recombination sites BBa_I11022 and BBa_I11023.

The following is excerpted from Radman-Livaja et al. RadmanLivaja and Landy et al. Landy. It has been edited for clarity.

Bacteriophage λ has long served as a model system for studies of regulated site-specific recombination. In conditions favorable for bacterial growth, the phage genome is inserted into the Escherichia coli genome by an ‘integrative’ recombination reaction, which takes place between DNA attachment sites called attP and attB in the phage and bacterial genomes, respectively. As a result, the integrated λ DNA is bounded by hybrid attachment sites, termed attL and attR. In response to the physiological state of the bacterial host or to DNA damage, λ phage DNA excises itself from the host chromosome. This excision reaction recombines attL with attR to precisely restore the attP and attB sites on the circular λ and E. coli DNAs Campbell. The phage-encoded λ integrase protein (Int), a tyrosine recombinase, splices together bacterial and phage attachment sites. Int is required for both integration and excision of the λ prophage Zissler67.

λ recombination has a strong directional bias in response to environmental conditions. Accessory factors, whose expression levels change in response to host physiology, control the action of Int and determine whether the phage genome will remain integrated or be excised. Int has two DNA-binding domains: a C-terminal domain, consisting of a catalytic domain and a core-binding (CB) domain, that interacts with the core recombining sites and an N-terminal domain (N-domain) that recognizes the regulatory arm DNA sites Wojciak02. The heterobivalent Int molecules bridge distant core and arm sites with the help of accessory proteins, such as integration host factor (IHF), which bend the DNA at intervening sites, and appose arm and core sequences for interaction with the Int recombinase. Five arm DNA sites in the regions flanking the core of attP are differentially occupied during integration and excision reactions. The integration products attL and attR cannot revert back to attP and attB without assistance from the phage-encoded factor Xis, which bends DNA on its own or in combination with the host-encoded factor Fis Abremski82,Abremski81,Hoess80,Thompson87a,Thompson87b,Ball91. Xis also inhibits integration, and prevents the attP and attB products of excision from reverting to attL and attR Abremski81,18]. Excision is inhibited by high concentrations of IHF <cite>Bushman85,Thompson86. Because the cellular levels of IHF and Fis proteins respond to growth conditions, these host-encoded factors have been proposed as the master signals for integration and excision Thompson87a,Bushman85,Thompson86, Nilsson92,Ball92, Ball91.

The following is excerpted from Landy et al. Landy. It has been edited for clarity.

The phage (attP) and bacterial (attB) att sites are designated POP’ and BOB’, respectively, and the prophage att sites are designated BOP’ (attL) and POB’ (attR). Transducing phage carrying attL (λgal) or attR (λbio) are generated from a prophage which has excised from the host chromosome by a rare int-independent recombination which deletes phage DNA from one end of the prophage and adds bacterial DNA to the other Campbell. A phage carrying the bacterial att site, BOB’, is obtained as a product of int-promoted recombination between a gal (BOP’) and a bio (POB’) transducing phage which is capable of transducing gal and bio together Echols70.

When Escherichia coli carries a deletion of the primary bacterial att site BOB’, int-dependent integration of λ can be detected at numerous loci (secondary bacterial att sites) on the E. coli chromosome Shimada72. This integration always involves the phage att site (POP’) Shimada75 and is thus very similar to the behavior of IS-elements Fiandt72, Hirsch72. The secondary prophage att sites are given the general designation ΔOP’ and POΔ’ and they differ from BOP’ and POB’ and from each other in their biological properties as determined by int- and xis -dependent recombination frequencies with various att sites.

References

<biblio>

1. Campbell Campbell, AM. Episomes. In: Caspari EW, Thoday JM. , editors. Advances in Genetics. 1. New York: Academic Press; 1962. pp. 101–145.
2. Zissler67 pmid=5637199
3. Guameros70 pmid=4907272
4. Kaiser70 pmid=4907271
5. Echols70 pmid=4907273
6. Shimada72 pmid=4552408
7. Shimada75 pmid=1095763
8. Fiandt72 pmid=4567155
9. Hirsch72 pmid=4567154
10. Hoess80 pmid=6446713
11. Abremski81 pmid=6279866
12. Abremski82 pmid=6213611
13. Bushman85 pmid=2932798
14. Thompson86 pmid=2946666
15. Thompson87a pmid=2957063
16. Thompson87b pmid=2958633
17. Kitts88a pmid=2970060
18. Kitts88b pmid=2975338
19. NunesDuby87 pmid=3040260
20. Holliday64 Holliday, R. A mechanism for gene conversion in fungi. Genet Res. 1964;5:282–304.
21. Ball91 pmid=1829453
22. Ball92 pmid=1459953
23. Nilsson92 pmid=1732224
24. Hallett97 pmid=9348666
25. Azaro02 Azaro, MA; Landy, A. λ Int and the λ Int family. In: Craig NL, Craigie R, Gellert M, Lambowitz A. , editors. Mobile DNA II. Washington, DC: ASM Press; 2002. pp. 118–148.
26. Wojciak02 pmid=11904406
27. Landy pmid=331474
28. Landy89 pmid=2528323
29. RadmanLivaja pmid=16368232

</biblio>

Bacteriophage P22 att DNA recombination system

The 2004 Boston University iGEM team designed and constructed the P22 att recombination sites BBa_I11032 and BBa_I11033.

The following text is excerpted from Cho et al. Cho99. It has been edited for clarity.

Bacteriophage P22 is a lambdoid phage which infects Salmonella typhimurium. P22 can integrate into and excise out of its host chromosome via site-specific recombination. Both integration and excision reactions require the phage-encoded int gene Smith67, and excision is dependent on the xis gene as well.

P22 Int is a member of the λ integrase family Argos86, Esposito97, NunesDuby98. The Int proteins of λ and P22 are composed of two domains. The catalytic domain binds to the core region of the phage recombination site, attP, where the actual recombination reactions occur. The smaller amino-terminal domain binds to arm-type sequences which are located on either site of the core within the attP Vargas88, Mungo94. The active components of λ integrative and excisive recombination are nucleosome-like structures, called intasomes, in which DNA is folded around several molecules of Int and integration host factor (IHF) Better82, Griffith85, Pollock83, Richet88, Robertson88. It has been demonstrated that one monomer of λ integrase can simultaneously occupy both a core-type binding site and an arm-type binding site Kim90, MacWilliams96. Formation of these bridges is facilitated by IHF, which binds to specific sequences and imparts a substantial bend to the DNA Craig84, Rice96, Robertson88, Snyder89.

The attP regions of P22 and λ are also similar in that both contain arm regions, known as the P and P′ arms, which contain Int arm-type binding sites and IHF binding sites Leong86, Mungo94. However, the arrangement, spacing, and orientation of the Int and IHF binding sites are distinct Mungo94. The attP region of λ contains two Int arm-type binding sites on the P arm and three on the P′ arm. The P arm contains two IHF binding sites, and the P′ arm contains a single site. The attP region of P22 contains three Int arm-type binding sites on the P arm and two sites on the P′ arm. In addition, IHF binding sites, called H and H′, are located on each arm of the P22 attP. Leong et al. Leong85 showed that the Escherichia coli IHF can recognize and bind to these P22 IHF binding sites in vitro. It was also shown that the maximum amount of P22 integrative recombination occurred in the presence of E. coli IHF in vitro, whereas in its absence, recombination was detectable but depressed Mungo94. However, the requirement for IHF or other possible accessory proteins during P22 site-specific recombination in vivo has not been tested. In this study, we assessed the role of IHF in P22 integration and excision in vivo.

Although the attP region of P22 contains strong IHF binding sites, in vivo measurements of integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid integration assay showed that integrative recombination occurs equally well in wild-type and ihfA mutant cells. P22 integrative recombination is also efficient in Escherichia coli in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another factor(s) can substitute for IHF in the formation of complexes.

References

<biblio>

1. Cho99 pmid=10400581
2. Smith67 Smith, H O; Levine, M. A phage P22 gene controlling integration of prophage. Virology. 1967;31:297–316.
3. Susskind78 pmid=353481
4. Leong86 pmid=3491212
5. Argos86 pmid=3011407
6. Esposito97 pmid=9278480
7. NunesDuby98 pmid=9421491
8. Vargas88 pmid=2843292
9. Mungo94 pmid=8051182
10. Better82 pmid=6310548
11. Griffith85 pmid=3159013
12. Pollock83 pmid=6226803
13. Richet88 pmid=2964274
14. Robertson88 pmid=2831189
15. Kim90 pmid=2146029
16. MacWilliams96 pmid=8807282
17. Craig84 pmid=6096022
18. Rice96 pmid=8980235
19. Snyder89 pmid=2528698
20. Leong85 pmid=2984205

</biblio>

=Saccharomyces cerevisiae-derived Flp/FRT DNA recombination system

Chris Anderson, a professor of bioengineering at UC Berkeley, constructed the FRT recombination site BBa_J61020.

Site-specific recombination provides a vehicle to introduce exogenous DNA, delete DNA, or rearrange DNA at specific sites in a chromosome (41). Among the site-specific recombination systems characterized to date, the FLP system of the yeast 2mm plasmid and the Cre-lox system of bacteriophage P1 are among the most attractive for genomic manipulation because of their efficiency, simplicity, and demonstrated in vivo activity in a wide range of organisms. These systems have been used to construct specific genomic deletions and gene duplications, study gene function, promote chromosomal translocations, promote site-specific chromosome cleavage, and facilitate the construction of genomic libraries in organisms including bacteria, yeast, insects, plants, mice, and humans (2–5, 10–18, 24–26, 28, 30–35, 38–41, 44, 45, 47, 50). These studies have only begun to tap the potential of the approach. Site-specific recombination catalyzed by the FLP and Cre recombinases occurs readily in bacterial cells (1, 5, 6, 21, 33). In principle, it could find wide application to studies of genomic structure and function as well as enhance the usefulness of Escherichia coli in biotechnology. Ironically, this approach has not been exploited in bacteria as it has been in eukaryotes, although bacteria were the first nonyeast cells in which FLP-mediated recombination was demonstrated (6). Even though gene targeting in bacteria can be achieved by homologous recombination, chromosomal targeting by sitespecific recombination provides a new route to stable transformation with the advantages of very high efficiency, defined reproducible insertion sites in the chromosome, and controlled reversibility. The yeast FLP system has been studied intensively (7, 8, 22, 36). The only requirements for FLP recombination are the FLP protein and the FLP recombination target (FRT) sites on the DNA substrates. The minimal functional FRT site contains only 34 bp. The FLP protein can promote both inter- and intramolecular recombination. Previously, we reported the construction of a model system in E. coli using the FLP recombination system for chromosomal targeting and demonstrated the effectiveness of the general approach (21). Site-specific integration was absolutely dependent upon the expression of FLP protein and the presence of FRT sites in the chromosome. In some experiments, from 1 to 10% of the exogenous DNA molecules used, introduced on a modified bacteriophage l vector, actually found their way into a cell and were integrated into the chromosome specifically at a chromosomal FRT.

References

<biblio>

1. Schweizer pmid=12736528
2. Huang pmid=9324255

</biblio>

E. coli XerCD/dif DNA recombination system

Xiaonan Wang, a member of the [http://parts.mit.edu/igem07/index.php/Edinburgh 2007 University of Edinburgh iGEM team], designed the dif recombination sites BBa_I742101 and BBa_I742102.

The following text is excerpted from Ip et al. Ip03.

The separation and segregation of newly replicated [E. coli] circular chromosomes can also be prevented by the formation of circular chromosome dimers, which can arise during crossing over by homologous recombination Blakely91; Clerget91; Kuempel91. In E. coli, these dimers, which arise about once every six generations, are resolved to monomers by the action of the FtsK–XerCD–dif chromosome dimer resolution machinery Steiner98a, Steiner98b, Recchia99, Steiner99. Two site-specific recombinases of the tyrosine recombinase family, XerCD, act at a 28 bp recombination site, dif, located in the replication terminus region of the E. coli chromosome to remove the crossover introduced by dimer formation, thereby converting dimers to monomers. A complete dimer resolution reaction during recombination at dif requires the action of the C-terminal domain of FtsK (FtsK_C) Steiner99; Barre00. FtsK is a multifunctional protein whose N-terminal domain acts in cell division, while the C-terminal domain functions in chromosome segregation Liu98; Wang98; Yu98a, Yu98b. Therefore, FtsK is well suited to coordinate chromosome segregation and cell division. A purified protein, FtsK_50C, containing a functional C-terminal domain, can translocate DNA in an ATP-dependent manner and activate Xer recombination at the recombination site dif, thereby reconstituting in vitro the expected in vivo activities of the C-terminal domain of the complete FtsK protein Aussel02.

References

<biblio>

1. Blakely91 pmid=1931824
2. Clerget91 pmid=1931823
3. Kuempel91 pmid=1657123
4. Steiner98a pmid=9484882
5. Steiner98b pmid=9829936
6. Liu98 pmid=9723927
7. Wang98 pmid=9723913
8. Yu98a pmid=9495771
9. Yu98b pmid=9829960
10. Steiner99 pmid=10027974
11. Recchia99 pmid=10523315
12. Barre00 pmid=11114887
13. Aussel02 pmid=11832210
14. Ip03 pmid=14633998

</biblio>