Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA

Pascal Gamas; Nancy L. Craig

doi:10.1093/nar/20.10.2525

Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA

Pascal Gamas, Nancy L. Craig

School of Medicine

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

31 Scopus citations

Abstract

The bacterial transposon Tn7 encodes five transposition genes tnsABCDE. We report a simple and rapid procedure for the purification of TnsC protein. We show that purified TnsC is active in and required for Tn7 transposition in a cell-free recombination system. This finding demonstrates that TnsC participates directly in Tn7 transposition and explains the requirement for tnsC function in Tn7 transposition. We have found that TnsC binds adenine nucleotides and is thus a likely site of action of the essential ATP cofactor in Tn7 transposition. We also report that TnsC binds non-specifically to DNA in the presence of ATP or the generally non-hydrolyzable analogues AMP-PNP and ATP-_γ-S, and that TnsC displays little affinity for DNA in the presence of ADP. We speculate that TnsC plays a central role in the selection of target DNA during Tn7 transposition.

Original language	English (US)
Pages (from-to)	2525-2532
Number of pages	8
Journal	Nucleic acids research
Volume	20
Issue number	10
DOIs	https://doi.org/10.1093/nar/20.10.2525
State	Published - May 25 1992

ASJC Scopus subject areas

Genetics

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title = "Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA",

abstract = "The bacterial transposon Tn7 encodes five transposition genes tnsABCDE. We report a simple and rapid procedure for the purification of TnsC protein. We show that purified TnsC is active in and required for Tn7 transposition in a cell-free recombination system. This finding demonstrates that TnsC participates directly in Tn7 transposition and explains the requirement for tnsC function in Tn7 transposition. We have found that TnsC binds adenine nucleotides and is thus a likely site of action of the essential ATP cofactor in Tn7 transposition. We also report that TnsC binds non-specifically to DNA in the presence of ATP or the generally non-hydrolyzable analogues AMP-PNP and ATP-γ-S, and that TnsC displays little affinity for DNA in the presence of ADP. We speculate that TnsC plays a central role in the selection of target DNA during Tn7 transposition.",

author = "Pascal Gamas and Craig, {Nancy L.}",

note = "Funding Information: We are particularly grateful to Roland Bainton for numerous helpful discussions. We thank Lidia Arciszewska and Ken Kubo for providing TnsB, TnsD and also helpful comments about protein purification, and Karina Orle for plasmids and antibodies. We also thank Ann Hagemann, Ken Kubo and Anne Stellwagen for comments on the manuscript. PG was supported the NIH-CNRS program and a Nato Fellowship. The work was supported by a grant from the National Institute of Allergy and Infectious Disease to NLC.",

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doi = "10.1093/nar/20.10.2525",

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T1 - Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA

AU - Gamas, Pascal

AU - Craig, Nancy L.

N1 - Funding Information: We are particularly grateful to Roland Bainton for numerous helpful discussions. We thank Lidia Arciszewska and Ken Kubo for providing TnsB, TnsD and also helpful comments about protein purification, and Karina Orle for plasmids and antibodies. We also thank Ann Hagemann, Ken Kubo and Anne Stellwagen for comments on the manuscript. PG was supported the NIH-CNRS program and a Nato Fellowship. The work was supported by a grant from the National Institute of Allergy and Infectious Disease to NLC.

PY - 1992/5/25

Y1 - 1992/5/25

N2 - The bacterial transposon Tn7 encodes five transposition genes tnsABCDE. We report a simple and rapid procedure for the purification of TnsC protein. We show that purified TnsC is active in and required for Tn7 transposition in a cell-free recombination system. This finding demonstrates that TnsC participates directly in Tn7 transposition and explains the requirement for tnsC function in Tn7 transposition. We have found that TnsC binds adenine nucleotides and is thus a likely site of action of the essential ATP cofactor in Tn7 transposition. We also report that TnsC binds non-specifically to DNA in the presence of ATP or the generally non-hydrolyzable analogues AMP-PNP and ATP-γ-S, and that TnsC displays little affinity for DNA in the presence of ADP. We speculate that TnsC plays a central role in the selection of target DNA during Tn7 transposition.

AB - The bacterial transposon Tn7 encodes five transposition genes tnsABCDE. We report a simple and rapid procedure for the purification of TnsC protein. We show that purified TnsC is active in and required for Tn7 transposition in a cell-free recombination system. This finding demonstrates that TnsC participates directly in Tn7 transposition and explains the requirement for tnsC function in Tn7 transposition. We have found that TnsC binds adenine nucleotides and is thus a likely site of action of the essential ATP cofactor in Tn7 transposition. We also report that TnsC binds non-specifically to DNA in the presence of ATP or the generally non-hydrolyzable analogues AMP-PNP and ATP-γ-S, and that TnsC displays little affinity for DNA in the presence of ADP. We speculate that TnsC plays a central role in the selection of target DNA during Tn7 transposition.

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Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA

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