Plasmid vectors for the analysis of protein-induced DNA bending

Christian Zwieb, Sankar Adhya

Research output: Chapter in Book/Report/Conference proceedingChapter

6 Scopus citations

Abstract

Bending is not only required to accommodate DNA within the cell but also is a mechanism used by proteins to initiate DNA replication, transcription, and recombination. Determining the angles by which regulatory DNA segments deviate from linearity upon binding of proteins is a necessary step toward a better understanding of a large number of essential biological functions. The pBend plasmids contain duplicate sets of restriction sites and, when combined with "gel shift" experiments, allow the straightforward determination of the bending angle in a DNA molecule. The steps for successfully carrying out a binding/bending experiment are described. They include the cloning of the protein-binding site into the chosen pBend vector, the isolation of a series of DNA fragments with identical in length but variable placing of the protein-binding site, and the gel electrophoretic analysis of the free and protein-bound fragments.

Original languageEnglish (US)
Title of host publicationDNA-Protein Interactions
Subtitle of host publicationPrinciples and Protocols, Third Edition
EditorsTom Moss, Benoit Leblanc
Pages547-562
Number of pages16
DOIs
StatePublished - Dec 1 2009
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume543
ISSN (Print)1064-3745

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Keywords

  • DNA bending
  • Protein-DNA complexes
  • Recombination
  • Replication
  • Transcription

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Zwieb, C., & Adhya, S. (2009). Plasmid vectors for the analysis of protein-induced DNA bending. In T. Moss, & B. Leblanc (Eds.), DNA-Protein Interactions: Principles and Protocols, Third Edition (pp. 547-562). (Methods in Molecular Biology; Vol. 543). https://doi.org/10.1007/978-1-60327-015-1_32