Identification of the Structural and Functional Domains of the Large Serine Recombinase TnpX from Clostridium perfringens

Abstract

Members of the large serine resolvase family of sitespecific recombinases are responsible for the movement of several mobile genetic elements; however, little is known regarding the structure or function of these proteins. TnpX is a serine recombinase that is responsible for the movement of the chloramphenicol resistance elements of the Tn4451/3 family. We have shown that TnpX binds differentially to its transposon and target sites, suggesting that resolvase-like excision and insertion were two distinct processes. To analyze the structural and functional domains of TnpX and, more specifically, to define the domains involved in protein-DNA and protein-protein interactions, we conducted limited proteolysis studies on the wild-type dimeric TnpX 1-707 protein and its functional truncation mutant, TnpX 1-597 . The results showed that TnpX was organized into three major domains: domain I (amino acids (aa) 1-170), which included the resolvase catalytic domain; domain II (aa 170 -266); and domain III (aa 267-707), which contained the dimerization region and two separate regions involved in binding to the DNA target. A small polypeptide (aa 533-587) was shown to bind specifically to the TnpX binding sites providing further evidence that it was the primary DNA binding region. In addition, a previously unidentified DNA binding site was shown to be located between residues 583 and 707. Finally, the DNA binding and multerimization but not the catalytic functions of TnpX could be reconstituted by recombining separate polypeptides that contain the N-and C-terminal regions of the protein. These data provide evidence that TnpX has separate catalytic, DNA binding, and multimerization domains.Mobile genetic elements, such as conjugative plasmids, transposons, integrons, and genomic islands are important vehicles for the transmission of virulence and antibiotic resistance genes in many microorganisms including Gram-positive bacteria such as Clostridium sp. (1, 2). Antibiotic resistance transposons identified in the clostridia include the integrative mobilizable elements Tn4451 and Tn4453a, which confer chloramphenicol resistance. Integration and excision of these elements is mediated by the large serine recombinase TnpX (3-5).Members of the serine recombinase family of site-specific recombinases catalyze strand exchange by a non-replicative DNA breakage and repair mechanism that involves a 2-bp staggered break across all four DNA strands and the formation of covalent phosphoserine linkages between the DNA strands and recombinase subunits (6). The large resolvases represent a subgroup within the serine recombinase family (7). The members of this subgroup are significantly larger (50 -82 kDa) than most other serine recombinase proteins (20 kDa) and catalyze a wider range of reactions (7). Little is known regarding the domain organization of large resolvases, and to date, the function and the mechanism by which the recombination process occurs is unclear.Unlike Tn3-like resolvase proteins that only catalyze excision reactions, large s...

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

“…Previous studies have shown that the aa 533-597 region of TnpX comprises an essential DNA binding site and that aa 598-707 contain a second non-essential binding region Lucet et al, 2005). As a result of an extensive random and site-directed mutagenesis study we have now identified a third region that is involved in DNA binding, aa 243-261.…”

“…Previous studies involved the construction of deletion derivatives of the tnpX gene based on bioinformatic and proteolytic analysis Lucet et al ., 2005). Our objective in this study was to isolate nonfunctional tnpX point mutants without any prior bias based on computer predictions of its structure.…”

“…To investigate the effect of mutation in the 243-261 region on any potential interaction with the non-essential C-terminal DNA binding region (Lucet et al, 2005), the C- 6. Alignment of the TnpX aa 236-263 region with other large serine recombinases.…”

“…The primary DNA binding region, aa 533-597, is essential for biological function . A truncated TnpX 533-597 protein will bind to its DNA target specifically but the larger TnpX 267-597 protein does not bind to DNA in the absence of the 1-266 region of TnpX (Lucet et al ., 2005). The second DNA binding region, aa 598-707, is not essential for biological activity as the TnpX 1-597 protein is functional .…”

“…Domain I contains the catalytic active site and domain III includes two distinct and independent DNAbinding motifs ( Fig. 1) (Lucet et al ., 2005). The primary DNA binding region, aa 533-597, is essential for biological function .…”