Faculty Publications

High-Resolution Characterization of DNA/Protein Complexes in Living Bacteria

Document Type

Article

Keywords

Architectural proteins, Chromatin endogenous cleavage (ChEC), Chromatin immunoprecipitation (ChIP), High-resolution mapping, Lac repression loop, Ligation-mediated PCR (LM-PCR), Phage lambda exonuclease, Polymerase chain reaction (PCR), Southern blot

Journal/Book/Conference Title

Methods in Molecular Biology

Volume

1837

First Page

95

Last Page

115

Abstract

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as the Escherichia coli lactose (lac) operon. Here, we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.

Department

Department of Chemistry and Biochemistry

Original Publication Date

1-1-2018

DOI of published version

10.1007/978-1-4939-8675-0_6

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