The IS<i>4</i> family of insertion sequences: evidence for a conserved transposase motif

Rezsöhazy, René; Hallet, Bernard; Delcour, Jean; Mahillon, Jacques

doi:10.1111/j.1365-2958.1993.tb01258.x

Cited by 149 publications

(144 citation statements)

References 64 publications

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“…Studies performed on HIV integrase have demonstrated that these two basic residues are important for catalytic activity and can be cross-linked to DNA ends (12). The DDE glutamate residue and the second lysine in Tnp make up part of the YREK signature motif that is conserved at the primary sequence level among all members of the IS4 family of transposases (including Tn5) (13). Data from various in vivo and in vitro assays of mutants in this region have been reported for transposase proteins from Tn10 (14 -17) and Tn903 (18).…”

Section: Tn5 Transposase (Tnp)mentioning

confidence: 99%

Tn 5 Transposase Active Site Mutants

Naumann

Reznikoff

2002

Journal of Biological Chemistry

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Tn5 transposase (Tnp) is a 53.3-kDa protein that is encoded by and facilitates movement of transposon Tn5. Tnp monomers contain a single active site that is responsible for catalyzing a series of four DNA breaking/ joining reactions at one transposon end. Based on primary sequence homology and protein structural information, we designed and constructed a series of plasmids that encode for Tnps containing active site mutations. Following Tnp expression and purification, the active site mutants were tested for their ability to form protein-DNA complexes and perform each of the four catalytic steps in the transposition pathway in vitro. The results demonstrate that Asp-97, Asp-188, and Glu-326, visible in the active site of Tn5 crystal structures, are absolutely required for all catalytic steps. Mutations within a series of amino acid residues that are conserved in the IS4 family of transposases and retroviral integrases also impair Tnp catalytic activity. Mutations at either Tyr-319 or Arg-322 reduce both hairpin resolution and strand transfer activity within protein-DNA complexes. Mutations at Lys-333 reduce the ability of Tnps to form protein-DNA complexes, whereas mutations at the less strongly conserved Lys-330 have less of an effect on both synaptic complex formation and catalytic activity.

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Section: Tn5 Transposase (Tnp)mentioning

confidence: 99%

Tn 5 Transposase Active Site Mutants

Naumann

Reznikoff

2002

Journal of Biological Chemistry

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“…These proteins have in common a conserved amino acid motif of two aspartates and a glutamate (known as the DDE or D,D35E motif) that is essential for catalytic activity (Kulkosky et al, 1992;Polard and Chandler, 1995a;Grindley and Leschziner, 1995). Other transposons, both bacterial and eukaryotic, also encode transposase proteins with similar, although less obviously related, features (Rezsohazy et al, 1993;Doak et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway

Lewis

Grindley

1997

Molecular Microbiology

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SummaryThe Escherichia coli insertion sequence, IS2, is a member of the IS3 family of bacterial transposable elements. Its transposase is a fusion protein, OrfAB, made by a programmed ¹1 translational frameshift near to the end of orfA and just after the start of orfB. We have characterized two major products of IS2 intramolecular transposition, which accumulate in cells that express the IS2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the complete IS2 with just a single basepair (occasionally 2 bp) separating the two IS ends. In all cases, this basepair is derived from the vector sequence immediately adjacent to the left IS2 end (IRL). The second product is a figure-eight molecule that contains all the IS2 and vector sequences present in the parental plasmid. One DNA strand contains the parental sequences unrearranged. The other contains a single-stranded version of the minicircle junction -the precise 3Ј end of IRR has been cleaved and joined to a target just outside the 5Ј end of IRL; the remaining vector sequences have a free 5Ј end, derived from cleavage at the 3Ј end of IRR, and a free 3Ј end, released upon cleavage of the target site adjacent to IRL. We propose that figure-eight molecules are the precursor to IS2 minicircles and that the formation of these two products is the initial step in IS2 intermolecular transposition. This proposed transposition pathway provides a means for a transposase that can cleave only one strand at each IS end to produce simple insertions and avoid forming co-integrates.

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“…1A) at the penultimate position on the nontransferred strand (27,28,32). The hairpin binding domain also contains the conserved Y-(2)-R-(3)-E-(6)-K signature found in the transposases of IS4 family members (33). This region of the transposase is involved in a series of interactions with the ends of the transposon DNA that distort the DNA and facilitate hairpin formation (Fig.…”

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confidence: 99%

Mixing active-site components: A recipe for the unique enzymatic activity of a telomere resolvase

Bankhead

Chaconas

2004

Proc. Natl. Acad. Sci. U.S.A.

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The ResT protein, a telomere resolvase from Borrelia burgdorferi, processes replication intermediates into linear replicons with hairpin ends by using a catalytic mechanism similar to that for tyrosine recombinases and type IB topoisomerases. We have identified in ResT a hairpin binding region typically found in cut-and-paste transposases. We show that substitution of residues within this region results in a decreased ability of these mutants to catalyze telomere resolution. However, the mutants are capable of resolving heteroduplex DNA substrates designed to allow spontaneous destabilization and prehairpin formation. These findings support the existence of a hairpin binding region in ResT, the only known occurrence outside a transposase. The combination of transposaselike and tyrosine-recombinase-like domains found in ResT indicates the use of a composite active site and helps explain the unique breakage-and-reunion reaction observed with this protein. Comparison of the ResT sequence with other known telomere resolvases suggests that a hairpin binding motif is a common feature in this class of enzyme; the sequence motif also appears in the RAG recombinases. Finally, our data support a mechanism of action whereby ResT induces prehairpin formation before the DNA cleavage step.

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The IS4 family of insertion sequences: evidence for a conserved transposase motif

Cited by 149 publications

References 64 publications

Tn 5 Transposase Active Site Mutants

Tn 5 Transposase Active Site Mutants

Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway

Mixing active-site components: A recipe for the unique enzymatic activity of a telomere resolvase

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