Two distinct regions of the large serine recombinase TnpX are required for DNA binding and biological function

Abstract

SummaryThe large serine recombinase, TnpX, from the Clostridium perfringens integrative mobilizable element Tn 4451 , consists of three domains and has two known DNA binding regions. In this study random and site-directed mutagenesis was used to identify other regions of TnpX that were required for biological activity. Genetic and biochemical analysis of these mutants led to the identification of important TnpX residues in the N-terminal catalytic pocket. In addition, another region of TnpX (aa 243-261), which is conserved within large serine recombinases, was shown to be essential for both excision and insertion. Mutation of charged residues within this region led to a loss of biological activity and aberrant DNA binding. This phenotype was mediated by interaction with the distal DNA binding region (aa 598-707). In these mutants, removal of residues 598-707 resulted in loss of DNA binding, despite the presence of the primary DNA binding region (aa 533-583). Analysis of mutations within the aa 243-261 region indicated that different protein conformations were involved in the insertion and the excision reactions. In summary, we have shown that TnpX is a complex protein that has multiple intra-and intermolecular interaction sites, providing insight into the structural and functional complexity of this important enzyme family.

Sequences in attB that affect the ability of ϕC31 integrase to synapse and to activate DNA cleavage

“…The geometry of DNA-binding is such that the C-terminal domain of resolvase extends around the DNA and contacts on the opposite side of the DNA to the catalytic serine (25). As in Bxb1 and TnpX, ϕC31 integrase has a proteolytically sensitive site between the N and C terminal domains (K152, unpublished data)(26,30,46). Moreover, the C-terminal domains of Bxb1 and TnpX have been shown previously to be capable of binding specifically to DNA (26,30,46).…”

“…As in Bxb1 and TnpX, ϕC31 integrase has a proteolytically sensitive site between the N and C terminal domains (K152, unpublished data)(26,30,46). Moreover, the C-terminal domains of Bxb1 and TnpX have been shown previously to be capable of binding specifically to DNA (26,30,46). Thus we propose that the C-terminal domains interact with the outer flanks of the att sites, that these interactions determine the conformations of integrase bound to each site and therefore whether they are compatible for synapsis.…”

Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

“…In a previous study, we cloned P attCI into the promoter probe vector pCB182, constructing pJIR1835 (14). To introduce TnpX into this system, we used pJIR2085, which contains a tnpX S15L allele, the product of which is catalytically inert (24), as the use of a plasmid expressing wild-type TnpX in E. coli resulted in the integration of the plasmid into the E. coli chromosome, disrupting P attCI (data not shown). The two plasmids pJIR1835 and pJIR2085 were introduced into E. coli CB454 (17), and ␤-galactosidase assays were performed as described previously (14,27).…”

Revised nomenclature for transposable genetic elements

“…1989; Boccard et al , 1989; Doublet et al , 2005). Similarly, both conjugative (Tn 5397 ) and mobilisable (Tn 4451 ) transposons encode serine recombinases that are responsible for their insertion and excision (Wang and Mullany, 2000; Adams et al , 2004; Adams et al , 2006). Many tyrosine and serine recombinases have been shown to require accessory factors to control the directionality of the recombination reactions they catalyze (insertion versus excision) (Lewis and Hatfull, 2001) however this is not true of all recombinases e.g.…”