The C-terminal α Helix of Tn5 Transposase Is Required for Synaptic Complex Formation

Steiniger-White, Mindy; Reznikoff, William S.

doi:10.1074/jbc.m003411200

Cited by 24 publications

(23 citation statements)

References 25 publications

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“…2 This combination of Tnp EK/LP and ME-flanked DNAs has resulted in the development of an efficient series of gel shift assays (4) and catalytic assays (2,23) used to study the transposition process. Tn5 in vitro assays have also been used to study C-terminally truncated forms of Tnp EK/LP and fulllength Tnp EK/LP with point mutations added to either the N or C terminus (26,27).…”

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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“…The concern was that inactivated deleted sequences could accumulate mutations altering functionally important homologies. N-terminal sequences starting at residue 26 are known to play a critical role in the required transposon DNA binding step (30), and Cterminal sequences, including residue 468 (out of 476), are known to be required for the essential transposase-transposase dimerization step (36).…”

Section: Resultsmentioning

confidence: 99%

Comparative Sequence Analysis of IS50/Tn5Transposase

2004

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Comparative sequence analysis of IS50 transposase-related protein sequences in conjunction with known structural, biochemical, and genetic data was used to determine domains and residues that play key roles in IS50 transposase function. BLAST and ClustalW analyses have been used to find and analyze six complete protein sequences that are related to the IS50 transposase. The protein sequence identity of these six homologs ranged from 25 to 55% in comparison to the IS50 transposase. Homologous motifs were found associated with each of the three catalytic residues. Residues that play roles in transposase-DNA binding, protein autoregulation, and DNA hairpin formation were also found to be conserved in addition to other residues of unknown function. On the other hand, some homologous sequences did not appear to be competent to encode the inhibitor regulatory protein. The results were also used to compare the IS50 transposase with the more distantly related transposase encoded by IS10.Transposable elements have played an important role in generating genetic diversity through their ability to cause insertions, deletions, inversions, and chromosome fusions. These elements are ubiquitous, and in some cases transposable elements and their inactive descendants can constitute the majority of a genome (12). The presence of a transposable element in a given genome is the product of lateral gene transfer. In bacteria, for instance, transposable elements may be introduced by means of DNA transformation, plasmid transfer, or phage infection. If an element was acquired recently by a given genome, it may well be absent from the genomes of close relatives.One class of transposable element ("cut and paste" transposons) moves from one DNA molecule to another (or from site to site within a DNA molecule) by a process that involves excision of the transposon DNA from the original site and integration of the transposon DNA into the second site. These DNA excision and integration processes are catalyzed by a transposon-specific protein called a transposase (26). Understanding how transposases function is of general importance because many transposases are members of a superfamily of proteins that includes human immunodeficiency virus type 1 integrase and possibly the RAG-1 protein responsible for the DNA cleavage events in the immune system V(D)J joining process. That is, this family of proteins share overall architectural properties, detailed active-site structures, and catalytic mechanisms (13,16,26,33).IS50 is a component of the composite transposon Tn5. IS50 transposase (also called Tn5 transposase) is an excellent model system because both biochemical and genetic data and detailed structural information are available (32, 33). As a result, the overall mechanism of IS50 transposition is well understood (see Fig. 1) and the roles of many protein residues in this process have been determined. However, structure-function studies of IS50 transposase are far from complete. For instance, the residues that participate in intramolecular regul...

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“…The papillation assay was The two bound transposase molecules form synaptic complexes in which the C-terminal ends of the two transposases dimerize (9). The catalytic domain of the transposase molecule bound to the left end is positioned to cleave the transposon end on the right end, and the transposase bound on the right end is positioned to cleave the left transposon end (10,21).…”

Section: Methodsmentioning

confidence: 99%

“…1A below) (8,9). Transposase cleaves the transposon from the donor backbone by a multistep reaction.…”

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confidence: 99%

Functional Characterization of Arginine 30, Lysine 40, and Arginine 62 in Tn5 Transposase

Twining

Goryshin

Bhasin

et al. 2001

Journal of Biological Chemistry

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Three N-terminal basic residues of Tn5 transposase, which are associated with proteolytic cleavages by Escherichia coli proteinases, were mutated to glutamine residues with the goal of producing more stable transposase molecules. Mutation of either arginine 30 or arginine 62 to glutamine produced transposase molecules that were more stable toward E. coli proteinases than the parent hyperactive Tn5 transposase, however, they were inactive in vivo. In vitro analysis revealed these mutants were inactive, because both Arg 30 and Arg 62 are required for formation of the paired ends complexes when the transposon is attached to the donor backbone. These results suggest Arg 30 and Arg 62 play critical roles in DNA binding and/or synaptic complex formation. Mutation of lysine 40 to glutamine did not increase the overall stability of the transposase to E. coli proteinases. This mutant transposase was only about 1% as active as the parent hyperactive transposase in vivo; however, it retained nearly full activity in vitro. These results suggest that lysine 40 is important for a step in the transposition mechanism that is bypassed in the in vitro assay system, such as the removal of the transposase molecule from DNA following strand transfer.

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The C-terminal α Helix of Tn5 Transposase Is Required for Synaptic Complex Formation

Cited by 24 publications

References 25 publications

Tn 5 Transposase Active Site Mutants

Tn 5 Transposase Active Site Mutants

Comparative Sequence Analysis of IS50/Tn5Transposase

Functional Characterization of Arginine 30, Lysine 40, and Arginine 62 in Tn5 Transposase

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