Isolation and Characterization of IS10Transposase Separation of Function Mutants: Identification of Amino Acid Residues in Transposase that are Important for Active Site Function and the Stability of Transposition Intermediates

Kennedy, Angela K; Haniford, David B.

doi:10.1006/jmbi.1996.0106

Cited by 23 publications

(20 citation statements)

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“…In the case of Tn10 Tnp, a mutation of the Lys-330 analog (Arg-297) was isolated as a dominant negative mutant (16). This is consistent with our results suggesting that substitution at this position may inhibit Tnp function.…”

Section: Tn5 Active Site Mutantssupporting

confidence: 90%

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 Active Site Mutantssupporting

confidence: 90%

Tn 5 Transposase Active Site Mutants

Naumann

Reznikoff

2002

Journal of Biological Chemistry

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“…Comparative rates of transposition were determined by measuring the relative ability of the transposases to incorporate a radiolabeled DNA fragment containing the left ITR of Himar1 into an unlabeled supercoiled plasmid target in a reaction similar to that for Tn10 in vitro (Fig. 4) (34). The fragment containing the ITR was labeled by cutting p27fH5Ј (10) with EcoRI and isolating the 111-bp fragment on a 1.5%, 1ϫ TAE agarose gel.…”

Section: Methodsmentioning

confidence: 99%

Hyperactive transposase mutants of the Himar 1 mariner transposon

Lampe

Akerley

Rubín

et al. 1999

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

301

271

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Mariner-family transposable elements are active in a wide variety of organisms and are becoming increasingly important genetic tools in species lacking sophisticated genetics. The Himar1 element, isolated from the horn fly, Haematobia irritans, is active in Escherichia coli when expressed appropriately. We used this fact to devise a genetic screen for hyperactive mutants of Himar1 transposase that enhance overall transposition from Ϸ4-to 50-fold as measured in an E. coli assay. Purified mutant transposases retain their hyperactivity, although to a lesser degree, in an in vitro transposition assay. Mutants like those described herein should enable sophisticated analysis of the biochemistry of mariner transposition and should improve the use of these elements as genetic tools, both in vivo and in vitro.

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“…It is noteworthy that mutants of IS10 transposase with a phenotype similar to that of S723A and S723C have been identified (Kennedy and Haniford 1996). These mutants exhibit rather minor cleavage defects in vitro, but fail to accumulate transposition products or cleaved intermediates in vivo.…”

Section: Postcleavage Defects End Release Errors and Chromosomal Trmentioning

confidence: 99%

“…These mutants exhibit rather minor cleavage defects in vitro, but fail to accumulate transposition products or cleaved intermediates in vivo. Importantly, these mutants, like the S723 mutant RAG1 proteins, generate unstable postcleavage complexes in vitro, leading to the proposal that cleaved intermediates fail to accumulate in vivo because of rapid breakdown of postcleavage complexes with attendant degradation of the cleaved DNA (Kennedy and Haniford 1996). Very similar processes may occur in vivo with the S723 mutant RAG1 proteins.…”

Section: Postcleavage Defects End Release Errors and Chromosomal Trmentioning

confidence: 99%

Evidence of a critical architectural function for the RAG proteins in end processing, protection, and joining in V(D)J recombination

Tsai

Drejer²,

Schatz³

2002

Genes Dev.

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In addition to creating the DNA double strand breaks that initiate V(D)J recombination, the RAG proteins are thought to play a critical role in the joining phase of the reaction. One such role, suggested by in vitro studies, might be to ensure the structural integrity of postcleavage complexes, but the significance of such a function in vivo is unknown. We have identified RAG1 mutants that are proficient in DNA cleavage but defective in their ability to interact with coding ends after cleavage and in the capture of target DNA for transposition. As a result, these mutants exhibit severe defects in hybrid joint formation, hairpin coding end opening, and transposition in vitro The variable portion of the genes encoding immunoglobulins and T cell receptors are assembled from component V, D, and J DNA segments by a site-specific recombination reaction termed V(D)J recombination (Tonegawa 1983). V(D)J recombination is targeted to specific sites on the chromosome by recombination signal sequences (RSSs) that flank antigen receptor gene segments. The RSS consists of a conserved heptamer (consensus, 5Ј-CACAGTG-3Ј) and nonamer (consensus, 5Ј-ACAAAAACC-

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Isolation and Characterization of IS10Transposase Separation of Function Mutants: Identification of Amino Acid Residues in Transposase that are Important for Active Site Function and the Stability of Transposition Intermediates

Cited by 23 publications

References 0 publications

Tn 5 Transposase Active Site Mutants

Tn 5 Transposase Active Site Mutants

Hyperactive transposase mutants of the Himar 1 mariner transposon

Evidence of a critical architectural function for the RAG proteins in end processing, protection, and joining in V(D)J recombination

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