DNA recombinase

More recently, a model that lacks functional T and B cells (SCID mice) has been described. SCID mice have defective DNA recombinase that results in an inability to make functional antigen receptors (Kotloff et al., 1993 Lpvik, 1995). As shown in Table 10.1-5, SCID mice have been used in a variety of test systems where a profound immunodeficiency is required. 

Cre is a bacterial recombinase (cre=causes recombination), which recognizes loxP sites of bacteriophage P. If two loxP (loxP= locus of x-ing over of bacteriophage P) sites have a parallel orientation, the DNA segment between these sites will be deleted by the action of the Cre recombinase. 

Classical global knockouts may have a developmental or lethal phenotype and thus preclude the analysis of the phenotypic consequences of the lack of a gene in specific tissues in adult animals. With the development of the cre/loxP and flp/FRT systems, it has become possible to excise defined DNA fragments from the genome of specified cells. Cre and Flp are bacterial and yeast recombinases, respectively, which recognize loxP and FRT sequences, respectively. The most common... 

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... 

Delete the lox-STOP-lox cassette in vitro by incubating 1 pg conditional shRNA plasmid DNA with 4 units of Cre recom-binase (New England Biolabs) in a total volume of 50 pi lx Cre buffer at 37°C for 30 min. Inactivate the recombinase for 10 min at 70°C. Eliminate nonrecombined plasmids by EcoRI digestion (see Note 8). 

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. 

Regulation by Recombination In the phase variation system of Salmonella, what would happen to the cell if the Hin recombinase became more active and promoted recombination (DNA inversion) several times in each cell generation ... 

Tyrosine recombinases of the lambda family also function in eukaryotes. Best known is the FLP (Flip) recombinase, which is encoded by the 2-pm plasmid of Saccharomyces cerevisiae and is thought to function in amplifying the number of plasmid copies.265 The 6.3-kbp plasmid contains a unique DNA sequence that lies between two 599-bp repeats in inverted orientation. Embedded in each repeat is an FLP recombination target (FRT) sequence, which is recognized by the plasmid recombinase. Each FRT segment includes inverted repeats 13 bp in length with an 8-bp spacer between them. As with other integrase systems the... 

The resolvase / invertase family and invertible DNA sequences. A second large family of recombinases act by cleaving a target DNA sequence hydrolytically leaving a free 3 -OH end (Eq. 27-14, step a). This free end then attacks a phosphodiester linkage in a second strand of DNA, cleaving that strand with an in-line nucleophilic displacement (step b). Active sites usually contain a characteristic cluster of aspartate and... 

Two other proteins are required for efficient inversion by the Hin recombinase. A dimer of a 98-residue helix-turn-helix DNA binding protein called Fis (factor for inversion stimulation), a relative of protein HU,18b must bind to an enhancer, a 65-bp DNA segment. Binding of Fis to the enhancer helps to hold the supercoiled DNA and the recombinase in a correct orientation for reaction.576 595 596 Protein HU is also needed. The same Fis protein binds to an enhancer... 

Microorganisms sometimes control the synthesis of surface proteins using segments of invertible DNA. The pathogenic bacterium Campylobacter fetus utilizes DNA rearrangements to allow one of a large family of surface layer (S-layer) proteins to be formed.597 The yeast FLP recombinase, mentioned in the preceding section, also inverts the sequence flanked by the 599-bp repeats.589... 

Figure 27-29 Formation of a synaptic complex of a super-coiled circular DNA containing the sites gix (green), which pass over and under the enhancer (gray). The recombinase Gin and the enhancer-binding Fis form a synaptic complex with DNA in this form as seen directly by electron microscopy. From Sadowski.576...

Replicative transposons. In 1979 Shapiro proposed the mechanism illustrated in Fig. 27-31 for replicative transposons. The two inversely repeated segments (green) at the ends of the transposon are aligned with the recipient DNA whose ends are labeled C and D. In fact, the recombining DNA molecules must be supercoiled.1 613 Staggered cuts are made in the recipient DNA at points a and b, which are 5, 9, or 11 bp apart, depending upon the specific recombinase. Nicks are also made in the transposon ends. The 3 ends from the transposon are resealed... 

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