title: 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 site-specific 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.
authors.affiliation: Department of Microbiology, Australian Bacterial Pathogenesis Program, Monash University, Victoria 3800, Australia.
authors.forename: Isabelle S
authors.initials: IS
authors.lastname: Lucet
authors.forename: Fleur E
authors.initials: FE
authors.lastname: Tynan
authors.forename: Vicki
authors.initials: V
authors.lastname: Adams
authors.forename: Jamie
authors.initials: J
authors.lastname: Rossjohn
authors.forename: Dena
authors.initials: D
authors.lastname: Lyras
authors.forename: Julian I
authors.initials: JI
authors.lastname: Rood
mesh_terms: D001426:Bacterial Proteins; D015139:Blotting, Southern; D015153:Blotting, Western; D020134:Catalytic Domain; D002918:Chymotrypsin / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000502:physiology*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D045522:Recombinases / Q000737:chemistry* / Q000502:physiology; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D045522:Recombinases; C497158:TnpX protein, Clostridium perfringens; D004247:DNA; D004254:DNA Nucleotidyltransferases; D002918:Chymotrypsin