DNA Footprinting: Method and Types

DNA Footprinting Overview

• DNA footprinting is a powerful method used for mapping where and how proteins bind tightly to DNA

History

• DNA footprinting was developed by David Galas and Albert Schmilz in 1978
• It was initially used to study the binding of the lac repressor
• The technique is a modification of the Maxam-Gilbert chemical sequencing technique

Principle

• DNA footprinting is an assay used to identify the site of protein-DNA interaction
• Proteins bound to DNA protect it from the action of degradative enzymes
• Primary application is to find the binding regions of DNA transcription factors

Procedure

• The DNA of interest is amplified using PCR
• The amplified double-stranded DNA is labeled at one end of each strand
• The protein of interest is added to the DNA
• The DNA is cleaved by a chemical or enzymatic cleavage agent
• The resulting fragments are run on a denaturing polyacrylamide gel

Types of DNA Footprinting

• There are two main types of DNA footprinting: DNAse I footprinting and DMS footprinting

DNAse I Footprinting

• DNAse I enzyme is utilized
• Helps find the target protein that binds to specific DNA and identify the sequence to which the protein is bound
• Protein binding to the DNA protects it from cleavage by DNAse I
• After cleavage, the remaining fragments are sequenced

DNAse I Footprinting Method

• End-labeled DNA is prepared
• The protein is bound to the DNA
• Mild digestion with DNAse I randomly cleaves double-stranded DNA on each strand
• DNA fragments are separated on denaturing acrylamide gels
• Samples in lanes will have increasing concentrations of DNA-binding protein

Dimethylsulfate (DMS) Footprinting

• DMS induces methylation of guanine residues
• Similar to DNAse footprinting
• DNA is mildly treated with DMS after protein addition, allowing for approximately one methylation per DNA molecule on average
• DNA is treated with a reagent to remove methylated purines
• Apurinic sites are removed using cleaving agents
• Each band ends next to a nucleotide that was methylated and thus unprotected by the protein

DMS Footprinting Method

• End-labelled DNA fragment is used
• Protein is bound
• It’s treated with DMS, which methylates purines
• Then partially cleaves DNA at methylated bases
• Fragments are separated on the gel.

DMS Footprinting Disadvantages

• Protein binding protects some purines from modification by DMS, but can stimulate modification of others, such as when the helix is distorted or partially melted
• Not suitable for detecting proteins binding to AT-rich sequences

Applications

• In vivo Footprinting: Used to analyze protein-DNA interactions occurring at a specific time in a cell
• Quantitative Footprinting: It is a modification of normal footprinting and is used to understand varying concentrations of proteins.
• Detection by capillary electrophoresis: Capillary electrophoresis devices are used.

In vivo Footprinting

• Analyzes protein-DNA interaction within a cell at a given time
• The cell membrane is permeabilized using UV rays
• DNAse I is utilized
• Following cleavage, single-stranded DNA is isolated, purified, and a linker DNA is added at the break points
• The region is amplified and run on a gel
• Coupled with immunoprecipitation, where the DNA-protein complex is precipitated with an antibody to the protein to allow for the assessment of specific protein binding regions

Quantitative Footprinting

• Modification of normal footprinting
• Varying protein concentrations are used
• Protein binding affinity can be estimated by observing footprinting with increasing protein concentration

Detection by Capillary Electrophoresis

• A capillary electrophoresis device is used
• PCR primers are coupled with carboxyflourescein, causing fragments produced by digestion to contain carboxyflourescein
• This fluorescence can be detected using a capillary electrophoresis device
• Transcription factor binding sites can be effectively identified