Mutagenesis of the IS1 transposase: importance of a His-Arg-Tyr triad for activity

Abstract

Inspection of the primary sequence of the IS1 transposase suggested that it carries residues which are characteristic of the active site of integrases of the bacteriophage family (Int). In particular, these include a highly conserved triad: His-Arg-Tyr. The properties of mutants made at each of these positions were investigated in vivo. The results of several different assays confirm that each is important for transposase activity. Moreover, as in the case of members of the Int family, different mutations of the His residue exhibited different effects. In particular, a His-to-Leu mutation resulted in complete inactivation whereas the equivalent His-to-Gln mutation retained low but significant levels of activity.

Presence of a Characteristic D-D-E Motif in IS1Transposase

“…All Int family proteins have histidine, arginine, and tyrosine residues constituting the H-R-Y motif in corresponding positions (1,3). However, these amino acid residues in IS1 transposase (H200, R203, and Y231 [44]) are not found in corresponding positions in transposases encoded by the other IS1 family elements. This suggests that these three amino acid residues do not constitute the active center of IS1 transposase.…”

“…Previously, IS1 transposase was thought to belong to the Int family with the H-R-Y motif (44). All Int family proteins have histidine, arginine, and tyrosine residues constituting the H-R-Y motif in corresponding positions (1,3).…”

Specific DNA cleavage mediated by the integrase of conjugative transposon Tn916

“…Some family members such as INT and FLP cleave DNA via the transient formation of a covalent protein-DNA linkage with the conserved Tyr residue (26,36), and mutation of this Tyr residue, as well as the other conserved residues, inhibits recombination (21,27,36,37,40). In contrast, IS1 transposase and the EcoRII restriction endonuclease have similar conserved residues that are required for enzymatic activity, yet these types of enzymes do not form covalent bonds with their DNA substrates (52,54). In vivo, the polarity of strand cleavage during Tn916 transposition is the same as that of other integrase family members (33).…”

ISBst12, a novel type of insertion-sequence element causing loss of S-layer-gene expression in Bacillus stearothermophilus ATCC 12980 The GenBank accession number for the sequence reported in this paper is AF162268.

“…Although members of the DDE family represent the majority of known IS elements, and mutagenic studies clearly underlined the importance of these residues, a significant fraction of IS elements does not exhibit a real or potential DDE triad. Interestingly, a highly conserved His-Arg-Tyr triad was identified in the putative transposase which resembles the signature of the catalytic site of integrases of the bacteriophage λ Int family and was also detected in the C-terminal part of the IS1 transposase (Abremski & Hoess, 1992 ;Argos et al, 1986 ;Serre et al, 1995). For the IS1 transposase it was demonstrated that each of the three amino acid residues of the conserved triad is important for transposase activity (Serre et al, 1995).…”

“…At the very C-terminal end of the protein, an acidic region with a calculated pI of 5n74 was detected (aa 401-482). Furthermore, an amino acid triad represented by His-276, Arg-279 and Tyr-308 was identified which resembles the signature of the highly conserved His-Arg-Tyr triad characteristic of the active site of integrases of the bacteriophage λ Int family (Figs 2 and 3) (Abremski & Hoess, 1992 ;Argos et al, 1986 ;Serre et al, 1995). The His-Arg-Tyr triad detected in the ISBst12 transposase was compared to the consensus sequence established for the active site of the integrase-family recombinases by Serre et al (1995), as shown in Fig.…”