Chimeric recombinases with designed DNA sequence recognition

Abstract

Site-specific recombination typically occurs only between DNA sequences that have co-evolved with a natural recombinase enzyme to optimize sequence recognition, catalytic efficiency, and regulation. Here, we show that the sequence recognition and the catalysis functions of a recombinase can be specified by unrelated protein domains. We describe chimeric recombinases with a catalytic domain from an activated multiple mutant of the bacterial enzyme Tn3 resolvase, fused to a DNA recognition domain from the mouse transcription factor Zif268. These proteins catalyze efficient recombination specifically at synthetic target sites recognized by two Zif268 domains. Our results demonstrate the functional autonomy of the resolvase catalytic domain and open the way to creating "custom-built" recombinases that act at chosen natural target sequences.

Fig. 1.

Site-specific recombination mediated by Tn3 resolvase. (A) Site-specific recombination reactions. A DNA segment containing a sequence of interest (arrow) may be excised by breaking and rejoining the DNA within two sites (boxes) recognized by a recombinase. (If the substrate is circular, this reaction is sometimes called resolution.) Similarly, the segment can be inserted by the intermolecular, reverse reaction. Another alternative, inversion of the orientation of the DNA segment between the sites, is not shown. (B) Crystal structure ofγδ resolvase dimer bound to a 34-bp DNA fragment containing res site I (10). The protein backbone is shown, with the N-terminal, catalytic domains in yellow, the C-terminal, DNA-binding domains in green, and the interdomain linkers in orange. The DNA is in gray spacefill representation. (C) Cartoon representation of the structure shown in B, using the same color scheme. The chimeric Z-resolvases described here have Zif268 DNA-binding domains in place of the resolvase C-terminal domains (DBD), and a mutant version of the catalytic domain (CAT). The Z-sites have motifs recognized by Zif268 in place of the motifs recognized by the resolvase C-terminal domain (indicated by arrows).

Fig. 2.

Structures of the substrates used to assay recombination in Escherichia coli.(Upper) pDB35 (15) contains two copies of site I, which are replaced by Z-sites in the other substrates. Km^r is a gene conferring kanamycin resistance. Recombination between the two sites divides the plasmid into two circles. The galK-containing circle is lost because it does not have an origin of replication or selective marker, making the cells galK^–, and giving pale yellow rather than red (galK⁺) colonies on the indicator plates. (Lower) Z-sites. Tn3 res site I sequence is red, the 9-bp Zif268-binding motif is blue, and the spacer sequence is white. The numbers indicate the lengths of the marked DNA segments (in base pairs). Arrows indicate the inverted repeats bound by recombinase (black for resolvase and unfilled for Z-resolvase).

Fig. 3.

Z-resolvase-mediated recombination in E. coli.(A) Expression plasmids encoding each resolvase variant were introduced into E. coli harboring plasmid substrates with either two copies of site I (Left) or two Z+6 sites (Right). The photographs show colonies on MacConkey indicator plates after 24 h. The percentage of plasmid molecules resolved was determined by isolating plasmid DNA from these cells (see Materials and Methods) and is given in the circle at the lower right of each panel (to the nearest whole number). S10A is a catalytically inactive mutant of Tn3 resolvase (control). NM is the N-terminal, catalytic domain of NM-resolvase (residues 1–148). NM-resolvase (NM-R) and Z-resolvase (Z-R) are described in Materials and Methods. The Z-resolvase used here has the L6 linker (C). (B) Assays of Z-resolvase (L6) activity on substrates containing Z-sites as shown in Fig. 2. The results are presented as in A. (C) Comparison of the activity of Z-resolvases with different linker sequences. The test substrate contained two Z+6 sites. Assays were performed as above; the percentage of resolved substrate molecules in the recovered plasmid DNA is presented on the right.

Fig. 4.

In vitro recombination by Z-resolvase (linker L6). (Left) Structure of the test substrate used, with two Z+6 sites in direct repeat. N denotes sites for the restriction enzyme NruI. (Right) Products after treatment of the substrate with Z-resolvase for 16 h were digested with NruI and separated on a 1.2% agarose gel. nr, nonrecombinant bands; r, recombinant bands. The sizes of the fragments (in base pairs) are indicated. Inversion gives 3,579- and 358-bp bands (the small fragment is not visible on the gel), whereas resolution gives 2,552- and 1,385-bp bands.