Recombination site-specific

An alternative to repeated cloning of PCR products is a recombination-based approach developed by Liu et al. (1998) to permit the cloning of a PCR product into a plasmid and the rapid conversion of the plasmid to a number of different expression systems without the necessity of cloning the PCR product multiple, independent times. The method, termed the univector plasmid-fusion system (UPS), involves the insertion of the PCR product into a particular type of plasmid, called the univector, which can then be placed under the control of a variety of promoters or fused in-frame to various tag sequences. The system is based upon plasmid fusion using the Cre-lox site-specific recombination system of bacteriophage PI (Sternberg et al., 1981). The Cre enzyme is a site-specific recombinase that catalyzes recombination between two 34 base pair (bp) loxP sequences and is involved in the resolution of dimers formed during replication of the... 

Figure 4.3. Univector plasmid fusion system. Cre- loxP mediated site-specific recombination fuses the pUNI and pHOST plasmids at the loxP site. As a result, the gene of interest is placed under the control of the pHOST promoter and fused to any Tag sequences present in the pHOST plasmid.

Abremski, K., Hoess, R., and Sternberg, N. (1983). Studies on the properties of PI site-specific recombination evidence for topologically unlinked products following recombination. Cell 32, 1301-1311. 

Hartley, J. L., Temple, G. F., and Brasch, M. A. (2000). DNA cloning using in vitro site-specific recombination. Genome Research 10, 1788-1795. 

Landy, A. (1989). Dynamic, structural, and regulatory aspects of site-specific recombination. Annu. Rev. Biochem. 55,913-949. 

Sternberg, N., Hamilton, D., Austin, S., Yarmolinsky, M., and Hoes, R. (1981). Site-specific recombination and its role in the life cycle of PI. Cold Spring Harbor Symp. Quant. Biol. 45, 297-309. 

Marker excision by site-specific recombination Very clean excision, small footprint Complex cloning procedure. Requires additional transgene encoding Cre recombinase 39... 

Abdel-Meguid, S. S., Grindley, N. D. F., Templeton, N. S. and Steitz, T. A. (1984). Qeavage of the site-specific recombination protein yS resolvase the smaller of the two fragments binds DNA specifically. Proc. Natl. Acad. Sci. USA 81,2001-2005. 

Gopaul, D. N., Guo, F. and Van Duyne, G. D. (1998). Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. EMBO J. 17, 4175-4187. 

DNA cloning using in vitro site-specific recombination. Grwowr 10, 1788-1795. 6... 

Corneille, S. et al. (2001). Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system. Plant. . 27 171-178. 

Encodes subunit of integration host factor (IHF), involved in site-specific recombination, replication, transposition, regulation of gene expression Required for recombinational repair... 

Site-Specific Recombination Results in Precise DNA Rearrangements... 

Homologous genetic recombination can involve any two homologous sequences. The second general type of recombination, site-specific recombination, is a very different type of process recombination is limited to spe-... 

FIGURE 25-39 Effects of site-specific recombination. The outcome of site-specific recombination depends on the location and orientation of the recombination sites (red and green) in a double-stranded DNA molecule. Orientation here (shown by arrowheads) refers to the order of nucleotides in the recombination site, not the 5 —>3 direction. 

The first site-specific recombination system studied in vitro was that encoded by bacteriophage A. When A phage DNA enters an E. coli cell, a complex series of regulatory events commits the DNA to one of two fates. 

Complete Chromosome Replication Can Require Site-Specific Recombination... 

Re combinational DNA repair of a circular bacterial chromosome, while essential, sometimes generates deleterious byproducts. The resolution of a Holliday junction at a replication fork by a nuclease such as RuvC, followed by completion of replication, can give rise to one of two products the usual two monomeric chromosomes or a contiguous dimeric chromosome (Fig. 25-41). In the latter case, the covalently linked chromosomes cannot be segregated to daughter cells at cell division and the dividing cells become stuck. A specialized site-specific recombination system in E. coli, the XerCD system, converts the dimeric chromosomes to monomeric chromosomes so that cell division can proceed. The reaction is a site-specific deletion reaction (Fig. 25-39b). This is another example of the close coordination between DNA recombination processes and other aspects of DNA metabolism. 

The recombination process is not as precise as the site-specific recombination described earlier, so additional variation occurs in the sequence at the V-J junction. This increases the overall variation by a factor of at least 2.5, thus the cells can generate about 2.5 X 1,200 = 3,000 different V-J combinations. The final joining of the V-J combination to the C region is accomplished by an RNA-splicing reaction after transcription, a process described in Chapter 26. 

Site-specific recombination occurs only at specific target sequences, and this process can also involve a Holliday intermediate. Recombinases cleave the DNA at specific points and ligate the strands to new partners. This type of recombination is found in virtually all cells, and its many functions include DNA integration and regulation of gene expression. 

Hallet, B. Sherratt, D.J. (1997) Transposition and site-specific recombination adapting DNA cut-and-paste mechanisms to a variety of genetic rearrangements. FEMS Microbiol. Rev. 21,... 

Van Duyne, G.D. (2001) A structural view of Cre-loxP site-specific recombination. Annu. Rev. Biophys. Biomol Struct. 30, 87-104. 

Holliday intermediates in homologous genetic recombination differ from their formation in site-specific recombination ... 

The switch is accomplished by periodic inversion of a segment of DNA containing the promoter for a flagellin gene. The inversion is a site-specific recombination reaction (see Fig. 25-39) mediated by the Hin re-combinase at specific 14 bp sequences (hix sequences)... 

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