Recombinase site-specific

Sanchez-Contreras et al. 2002), and is mediated by the site-specific recombinases Sss and XerD (Martmez-Granero et al. 2005, 2006). An analogous phenomenon occurred in Pseudomonas sp. strain PCL1171 in which mutations in the genes gacA and gacS were shown to be responsible (van den Broek et al. 2005). 

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... 

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

Yang, W. and Steitz, T. A. (1995). Crystal structure of the site-specific recombinase yS resolvase complexed with a 34 bp cleavage site. Cell 82,193-207. 

A widely used inducible system in the mouse is the bacteriophage Pl-derived Cre-LoxP system (25). The site-specific Cre recombinase recognizes specifically 34 bp loxP sites. Generating mice that harbor a DNA sequence flanked by two loxP sites and... 

Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage PI. Proc Natl Acad Sci USA 85 5166-5170... 

In a recently developed method, site-specific recombinases from bacteriophage or yeast have been used to introduce subtle mutations or deletions... 

Even more elegantly, site-specific recombinases may be used to structurally disrupt a gene which had previously been provided with recombinase-specific sites, for example targeted to intronic sites (see also Section 2.2.4). Deletion of flanked exons, and consequently gene inactivation, will however... 

To complete the reaction, the process must be repeated at a second point within each of the two recombination sites (steps (3) and (4)). In some systems, both strands of each recombination site are cut concurrently and rejoined to new partners without the Holliday intermediate. The exchange is always reciprocal and precise, regenerating the recombination sites when the reaction is complete. We can view a recombinase as a site-specific endonuclease and ligase in one package. 

The sequences of the recombination sites recognized by site-specific recombinases are partially asymmetric (nonpalindromic), and the two recombining sites align in the same orientation during the recombinase reaction. The outcome depends on the location and orientation of the recombination sites (Fig. 25-39). If the two sites are on the same DNA molecule, the reaction either inverts or deletes the intervening DNA, determined by whether the recombination sites have the opposite or the same... 

FIGURE 25-41 DNA deletion to undo a deleterious effect of re-combinational DNA repair. The resolution of a Holliday intermediate during recombinational DNA repair (if cut at the points indicated by red arrows) can generate a contiguous dimeric chromosome. A specialized site-specific recombinase in E. coli, XerCD, converts the dimer to monomers, allowing chromosome segregation and cell division to proceed. 

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. 

Branda, C.S. and Dymecki, S.M. (2004) Talking about a revolution the impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6, 7-28. 

O Gorman, S., Fox, D.T., and Wahl, G.M. (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251,1351-1355. 

The success of in vitro selection depends on the level of functional display but also principally on the quality and the diversity of the starting library. The attainable diversity can be significantly increased by taking advantage of site-specific in vivo recombination (see Section 5.4). In this case, the bacterial host must express the appropriate site-specific recombinase. 

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