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Mutants of Tn 3 resolvase which do not require accessory binding sites for recombination activity
Author(s) -
Arnold Patricia H.,
Blake David G.,
Grindley Nigel D.F.,
Boocock Martin R.,
Stark W.Marshall
Publication year - 1999
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/18.5.1407
Subject(s) - tn3 transposon , synapsis , biology , site specific recombination , binding site , mutant , recombination , recombinase , dna , microbiology and biotechnology , genetics , transposable element , gene
Tn 3 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 I×site 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.