STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE H19Y MUTATED PRIMARY DNA RECOGNITION SUBDOMAIN OF SLEEPING BEAUTY TRANSPOSASE

Search for this publication on Google Scholar
Leighton, G. (2017). STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE H19Y MUTATED PRIMARY DNA RECOGNITION SUBDOMAIN OF SLEEPING BEAUTY TRANSPOSASE. Unc Charlotte Electronic Theses And Dissertations.
Download PDF

Abstract

The Sleeping Beauty transposon system, consisting of the transposon DNA and the transposase enzyme, is a member of the Tc1/mariner family of DNA transposons. Although it is an important tool in genetic applications and has been adapted for human gene therapy, its molecular mechanism remains obscure. Here, we use an experimental biophysics approach to study the molecular mechanism of the Sleeping Beauty transposon. We investigate the folding of the specific DNA recognition subdomain of the Sleeping Beauty transposase, the PAI subdomain. We show that only the folded PAI binds to DNA, however the amount of unfolded conformation is significant at close to physiologic conditions. We identify amino acid substitutions that result in stabilization of its folded conformation. Overall, our results provide a mechanistic insight into DNA recognition by the Sleeping Beauty transposase and suggest modifications to improve its activity.

Details
Author

Leighton, Gage

Title

STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE H19Y MUTATED PRIMARY DNA RECOGNITION SUBDOMAIN OF SLEEPING BEAUTY TRANSPOSASE

Physical Description

1 online resource (40 pages) : PDF

Date

2017

Degree Granting Institution

University of North Carolina at Charlotte

Abstract

The Sleeping Beauty transposon system, consisting of the transposon DNA and the transposase enzyme, is a member of the Tc1/mariner family of DNA transposons. Although it is an important tool in genetic applications and has been adapted for human gene therapy, its molecular mechanism remains obscure. Here, we use an experimental biophysics approach to study the molecular mechanism of the Sleeping Beauty transposon. We investigate the folding of the specific DNA recognition subdomain of the Sleeping Beauty transposase, the PAI subdomain. We show that only the folded PAI binds to DNA, however the amount of unfolded conformation is significant at close to physiologic conditions. We identify amino acid substitutions that result in stabilization of its folded conformation. Overall, our results provide a mechanistic insight into DNA recognition by the Sleeping Beauty transposase and suggest modifications to improve its activity.

Genre

masters theses

Subjects--Topics

Physics
Chemistry, Physical and theoretical
Biophysics

Degree

M.S.

Keywords

DNA Binding
Gene Therapy
Hyperactive Mutation
PAI Subdomain
Sleeping Beauty Transposase
Transposition

Subject Area

Applied Physics

Advisor(s)

Nesmelova, Irina

Committee Members

Jacobs, Donald
Krueger, Joanna

Degree Note

Thesis (M.S.)--University of North Carolina at Charlotte, 2017.

Rights Statement

This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). For additional information, see http://rightsstatements.org/page/InC/1.0/.

Rights Holder Information

Copyright is held by the author unless otherwise indicated.

Identifier

Leighton_uncc_0694N_11516

Permalink

http://hdl.handle.net/20.500.13093/etd:258