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Activating mutations of Tn 3 resolvase marking interfaces important in recombination catalysis and its regulation
Author(s) -
Burke Mary E.,
Arnold Patricia H.,
He Jiuya,
Wenwieser Sandra V. C. T.,
Rowland SallyJ.,
Boocock Martin R.,
Stark W. Marshall
Publication year - 2004
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03831.x
Subject(s) - tn3 transposon , recombinase , site specific recombination , biology , mutant , tetramer , synapsis , plasmid , holliday junction , recombination , dna , mutation , genetics , binding site , dimer , transposable element , homologous recombination , biochemistry , chemistry , gene , enzyme , organic chemistry
Summary Catalysis of DNA recombination by Tn 3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites ( res ). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent ‘accessory’ binding sites II and III. ‘Hyperactive’ resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli . Some single mutants can resolve a res × site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I × site I plasmid. Site I × site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2–3′ interface that abolish activity of wild‐type resolvase. Activating mutations may allow regulatory mechanisms of the wild‐type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.