Defining characteristics of Tn5 Transposase non-specific DNA binding

Mindy Steiniger; Christian D. Adams; John F. Marko; William S. Reznikoff

doi:10.1093/nar/gkl179

Defining characteristics of Tn5 Transposase non-specific DNA binding

Mindy Steiniger, Christian D. Adams, John F. Marko, William S. Reznikoff^*

^*Corresponding author for this work

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

18 Scopus citations

Abstract

While non-specific DNA plays a role in target localization for many recombinases, transcription factors and restriction enzymes, the importance of non-specific DNA interactions for transposases has not been investigated. Here, we discuss non-specific DNA-Tn5 Transposase (Tnp) interactions and suggest how they stabilize the Tnp and modulate Tnp localization of the 19 bp Tnp recognition end sequences (ESes). DNA protection assays indicate that full-length Tnp interacts efficiently with supercoiled DNA that does not contain ESes. These interactions significantly prolong the lifetime of Tnp, in vitro. The balance between non-specific DNA bound and free Tnp is affected by DNA topology, yet, intermolecular transfer of active Tnp occurs with both supercoiled and linear non-specific DNA. Experiments with substrates of varying lengths show that Tn5 Tnp can utilize non-specific DNA to facilitate localization of an intramolecular ES over distances less than 464 bp. Finally, synaptic complex formation is inhibited in the presence of increasing concentrations of supercoiled and linear pUC19. These experiments strongly suggest that Tn5Tnp has a robust non-specific DNA binding activity, that non-specific DNA modulates ES sequence localization within the global DNA, most likely through a direct transfer mechanism, and that non-specific DNA binding may play a role in the cis bias manifested by Tn5 transposition.

Original language	English (US)
Pages (from-to)	2820-2832
Number of pages	13
Journal	Nucleic acids research
Volume	34
Issue number	9
DOIs	https://doi.org/10.1093/nar/gkl179
State	Published - 2006

Funding

The authors would like to thank Dr Ruth Saeker and Dr M. Thomas Record for helpful discussions and insight and Dr Brandon Ason and Dr Ambrose R. Kidd III for critical review of the manuscript. The authors would also like to thank Dr Dunja Skoko for assistance with the single DNA molecule experiments. This work was supported by NIH grant GM50692 to W.S.R. (M.S. and C.D.A) and by NSF grants DMR-0203963 and PHY-0445565, and by an award from the Focused Giving Program of Johnson & Johnson Corporation to J.F.M. (C.D.A.). Funding to pay the Open Access publication charges for this article was provided by NIGMS.

ASJC Scopus subject areas

Genetics

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10.1093/nar/gkl179

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title = "Defining characteristics of Tn5 Transposase non-specific DNA binding",

abstract = "While non-specific DNA plays a role in target localization for many recombinases, transcription factors and restriction enzymes, the importance of non-specific DNA interactions for transposases has not been investigated. Here, we discuss non-specific DNA-Tn5 Transposase (Tnp) interactions and suggest how they stabilize the Tnp and modulate Tnp localization of the 19 bp Tnp recognition end sequences (ESes). DNA protection assays indicate that full-length Tnp interacts efficiently with supercoiled DNA that does not contain ESes. These interactions significantly prolong the lifetime of Tnp, in vitro. The balance between non-specific DNA bound and free Tnp is affected by DNA topology, yet, intermolecular transfer of active Tnp occurs with both supercoiled and linear non-specific DNA. Experiments with substrates of varying lengths show that Tn5 Tnp can utilize non-specific DNA to facilitate localization of an intramolecular ES over distances less than 464 bp. Finally, synaptic complex formation is inhibited in the presence of increasing concentrations of supercoiled and linear pUC19. These experiments strongly suggest that Tn5Tnp has a robust non-specific DNA binding activity, that non-specific DNA modulates ES sequence localization within the global DNA, most likely through a direct transfer mechanism, and that non-specific DNA binding may play a role in the cis bias manifested by Tn5 transposition.",

author = "Mindy Steiniger and Adams, {Christian D.} and Marko, {John F.} and Reznikoff, {William S.}",

note = "Funding Information: The authors would like to thank Dr Ruth Saeker and Dr M. Thomas Record for helpful discussions and insight and Dr Brandon Ason and Dr Ambrose R. Kidd III for critical review of the manuscript. The authors would also like to thank Dr Dunja Skoko for assistance with the single DNA molecule experiments. This work was supported by NIH grant GM50692 to W.S.R. (M.S. and C.D.A) and by NSF grants DMR-0203963 and PHY-0445565, and by an award from the Focused Giving Program of Johnson & Johnson Corporation to J.F.M. (C.D.A.). Funding to pay the Open Access publication charges for this article was provided by NIGMS.",

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N1 - Funding Information: The authors would like to thank Dr Ruth Saeker and Dr M. Thomas Record for helpful discussions and insight and Dr Brandon Ason and Dr Ambrose R. Kidd III for critical review of the manuscript. The authors would also like to thank Dr Dunja Skoko for assistance with the single DNA molecule experiments. This work was supported by NIH grant GM50692 to W.S.R. (M.S. and C.D.A) and by NSF grants DMR-0203963 and PHY-0445565, and by an award from the Focused Giving Program of Johnson & Johnson Corporation to J.F.M. (C.D.A.). Funding to pay the Open Access publication charges for this article was provided by NIGMS.

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N2 - While non-specific DNA plays a role in target localization for many recombinases, transcription factors and restriction enzymes, the importance of non-specific DNA interactions for transposases has not been investigated. Here, we discuss non-specific DNA-Tn5 Transposase (Tnp) interactions and suggest how they stabilize the Tnp and modulate Tnp localization of the 19 bp Tnp recognition end sequences (ESes). DNA protection assays indicate that full-length Tnp interacts efficiently with supercoiled DNA that does not contain ESes. These interactions significantly prolong the lifetime of Tnp, in vitro. The balance between non-specific DNA bound and free Tnp is affected by DNA topology, yet, intermolecular transfer of active Tnp occurs with both supercoiled and linear non-specific DNA. Experiments with substrates of varying lengths show that Tn5 Tnp can utilize non-specific DNA to facilitate localization of an intramolecular ES over distances less than 464 bp. Finally, synaptic complex formation is inhibited in the presence of increasing concentrations of supercoiled and linear pUC19. These experiments strongly suggest that Tn5Tnp has a robust non-specific DNA binding activity, that non-specific DNA modulates ES sequence localization within the global DNA, most likely through a direct transfer mechanism, and that non-specific DNA binding may play a role in the cis bias manifested by Tn5 transposition.

AB - While non-specific DNA plays a role in target localization for many recombinases, transcription factors and restriction enzymes, the importance of non-specific DNA interactions for transposases has not been investigated. Here, we discuss non-specific DNA-Tn5 Transposase (Tnp) interactions and suggest how they stabilize the Tnp and modulate Tnp localization of the 19 bp Tnp recognition end sequences (ESes). DNA protection assays indicate that full-length Tnp interacts efficiently with supercoiled DNA that does not contain ESes. These interactions significantly prolong the lifetime of Tnp, in vitro. The balance between non-specific DNA bound and free Tnp is affected by DNA topology, yet, intermolecular transfer of active Tnp occurs with both supercoiled and linear non-specific DNA. Experiments with substrates of varying lengths show that Tn5 Tnp can utilize non-specific DNA to facilitate localization of an intramolecular ES over distances less than 464 bp. Finally, synaptic complex formation is inhibited in the presence of increasing concentrations of supercoiled and linear pUC19. These experiments strongly suggest that Tn5Tnp has a robust non-specific DNA binding activity, that non-specific DNA modulates ES sequence localization within the global DNA, most likely through a direct transfer mechanism, and that non-specific DNA binding may play a role in the cis bias manifested by Tn5 transposition.

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JF - Nucleic acids research

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ER -

Defining characteristics of Tn5 Transposase non-specific DNA binding

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