Site-specific recombinases

Both vectors enable the engineering of stable recombinant streptomycete strains without any selection marker. The availability of the marker-free expression system allows the generation of multiple, stable, unmarked integrations, and the expression of several genes simultaneously [25]. 

The same recombinase has been employed to excise an approximately 200 kb region from the chromosome of Streptomyces J1074 [46]. 

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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... 

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. 

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... 

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. 

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. 

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. 

Although the reactions catalyzed by the S- and Y- site-specific recombinases are weU characterized, little is known abont the biochemistry of their cousins, the Y- and S-transposases. These transposases are thonght to catalyze strand breakage and transfer as do their site-specific recombinase relatives. Neither of these requires divalent metal ions for catalysis. Althongh these transposases show site-specificity for the ends of the donor transposon, they seem more flexible than classic site-specific... 

Cre-ative duplexes. Site specific recombinases like Cre require that the sequence between the recombinase binding sites be identical in the two DNA molecules that are to be recombined. Examine the structures of the various Cre-DNA complexes in the Structural Insights module on Cre recombinase. How might the recombination mechanism check these sequences to ensure they are identical in the parent DNA molecules ... 

Carrasco, C.D., Ramaswamy, K.S., Ramasubramanian, T.S. and Golden J.W. (1994). Anabaena xisV gene encodes a developmental regulated site-specific recombinase. Genes Develop. 8, 74-83. 

In contrast to nucleic acid polymerases, polynucleotide processing enzymes often act by mechanisms that involve covalent polynucleotide enzymes as compulsory intermediates (53, 54). The covalent linkages are through phosphodies-ters comprising an enzymic nucleophile, usually the phenolic group of tyrosine, and a nucleotidyl moiety of the nucleic acid. These enzymes are not classified as nucleotidyltransferases, but they catalyze nucleotidyl group transfer as the basic reaction in isomerization processes. Examples are topoisomerases and site-specific recombinases. These enzymes utilize the enzymic nucleophile to cleave the polynucleotide in such a way as to preserve the energy of the covalent bond... 

Fig. 3.3-7 Site-specific recombinases can be used to control gene expression.

Poyart-Salmeron C, Trieu-Cuot P, Carlier C, Comvalin P (1989) Molecular characterization of two proteins involved in the excision of the conjugative transposon Tnl545 homologies with other site-specific recombinases. EMBO J 8 2425-2433... 

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