Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity

Abstract

Tn3 resolvase promotes site-specific recombination between two res sites, each of which has three resolvase dimer-binding sites. Catalysis of DNA-strand cleavage and rejoining occurs at binding site I, but binding sites II and III are required for recombination. We used an in vivo screen to detect resolvase mutants that were active on res sites with binding sites II and III deleted (that is, only site I remaining). Mutations of amino acids Asp102 (D102) or Met103 (M103) were sufficient to permit catalysis of recombination between site I and a full res, but not between two copies of site I. A double mutant resolvase, with a D102Y mutation and an additional activating mutation at Glu124 (E124Q), recombined substrates containing only two copies of site I, in vivo and in vitro. In these novel site Iϫsite I reactions, product topology is no longer restricted to the normal simple catenane, indicating synapsis by random collision. Furthermore, the mutants have lost the normal specificity for directly repeated sites and supercoiled substrates; that is, they promote recombination between pairs of res sites in linear molecules, or in inverted repeat in a supercoiled molecule, or in separate molecules.

Structure-guided reprogramming of serine recombinase DNA sequence specificity

“…1B). We introduced biased diversity into the arm regions of the accessory factor-independent mutants of Gin invertase (H106Y) and Tn3 resolvase (G70S, D102Y, and E124Q) by sitedirected saturation mutagenesis (33,34). The amino acid frequencies for each randomized position are available in Table S1.…”

Chimeric recombinases with designed DNA sequence recognition

“…The Z-site-containing test plasmids for the in vivo assays were constructed similarly to pDB35, a plasmid with two copies of Tn3 res site I (Fig. 2), which has been described (15). The substrates used for in vitro Z-site recombination assays were constructed similarly to pOG3, from the cloning vector pMTL23 (17).…”

“…The hyperactive NM-resolvase has the following six differences from the wild-type Tn3 resolvase sequence: R2A E56K G101S D102Y M103I Q105L. Zresolvase expression plasmids were constructed by cloning a fragment encoding the catalytic domain of NM-resolvase (residues 1-144), and synthetic fragments encoding the linker and the Zif268 DNA-binding domain, into a plasmid similar to pAT5 (15). The amino acid sequence of the 89-residue Zif268 DNA-binding domain was as described (16).…”

Synthesis of programmable integrases

“…The resulting chimera, Tn3C5, was expected to recombine C.20T (Table 1), a RecZF site containing the central 20 bp of the Tn3 native substrate (Fig. 6A) (10). Tn3C5-mediated integration in the 293-C.20T cell line occurred with high specificity of total integration (88.3 Ϯ 20.5%).…”

“…This intramolecular specificity is assured by obligate assembly of large protein complexes, wherein accessory factors bound at neighboring sites impose topological and spatial constraints on the recombination reaction (8). Hyperactive mutants of several serine resolvases and invertases have been discovered that efficiently catalyze unrestricted recombination between minimal dimer-binding sites (Table S1) (9,10). Furthermore, unlike other site-specific recombinases, serine resolvases and invertases are well suited to synthetic reengineering.…”