A Technique For Studies Dna Protein Interactions

Published Date: 02 Nov 2017

Protein–DNA interactions are when a protein binds a molecule of DNA, frequently to regulate the biological function of DNA, usually the expression of a gene. between the proteins that bind to DNA are transcription factors that activate or contain gene expression by binding to DNA motifs and histones that form part of the structure of DNA and attach to it less particularly. Also proteins that repair DNA such as uracil-DNA glycosylase interact closely with it.

In general, proteins bind to DNA in the major groove, however there are possibilities. Protein-DNA interaction are of mostly two types, 1)either specific interaction, 2)non-specific interaction.

Designing DNA-binding proteins that have a specified DNA-binding site has been an important objective for biotechnology. Zinc finger proteins have been designed to bind to specific DNA sequences and this is the foundation of zinc finger nucleases. Freshly transcription activator-like effector nucleases (TALENs) have been created which are based on natural proteins secreted by Xanthomonas bacteria by means of their type III secretion system when they infect various plant species.

Recognition methods

There are many techniques in vitro and in vivo which are useful in detecting DNA-Protein Interactions. The following lists some methods at present in use:

Electrophoretic mobility shift assay is a extensive technique to identify protein-DNA interactions.

DNase footprinting assay can be used to identify the specific site of binding of a protein to DNA.

Chromatin immunoprecipitation is used to identify the sequence of the DNA fragments which bind to a identified transcription factor. This technique when combined with high throughput sequencing is known as ChIP-Seq and when combined with microarrays it is known as ChIP-chip.

Yeast One-hybrid System (Y1H) is used to identify which protein binds to a particular DNA fragment.

Bacterial one-hybrid system (B1H) is used to identify which protein binds to a particular DNA fragment.

Structure determination using X-ray crystallography has been used to give a highly exhaustive atomic view of protein-DNA interactions.

DNA footprinting

DNA footprinting is a method of investigating the sequence specificity of DNA-binding proteins in vitro. This technique can be used to study protein-DNA interactions both outside and inside cells.

The regulation of transcription has been studied broadly, and however there is still much that is not known. Transcription factors and associated proteins that bind promoters, enhancers, or silencers to drive or contain transcription are essential to understanding the unique regulation of individual genes within the genome. Techniques like DNA footprinting will help make clear which proteins bind to these regions of DNA and unknot the complexities of transcriptional control.

Contents

1 History

2 Method

2.1 Labeling

2.2 Cleavage agent

3 Advanced Applications

3.1 In vivo foot printing

3.2 Quantitative foot printing

3.3 Foot printing coupled with detection by capillary electrophoresis

4 References

History

In 1978, David Galas and Albert Schmitz developed the DNA footprinting technique to study the binding specificity of the lac repressor protein. It was originally a modification of the Maxam-Gilbert chemical sequencing technique.

Method

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Figure 1. DNA footprinting workflow

The simplest application of this technique is to review whether a given protein binds to a region of interest inside a DNA molecule. The wet lab methodology is summarized, with suitable selection of reagents discussed, below.

Polymerase chain reaction (PCR). Amplify and label section of interest that contains a potential protein-binding site, ideally applicant is between 50 to 200 base pairs in length.

Add protein of interest to a portion of the labeled template DNA; a portion should stay separate without protein, for later contrast

Add a cleavage agent to both portions of DNA template. The cleavage agent is a chemical or enzyme that will cut at random locations in a sequence free manner. The reaction should occur just long sufficient to cut each DNA molecule in only one location. A protein that particularly binds a region within the DNA template will protect the DNA it is bound to from the cleavage agent.

Run both samples side by side on a polyacrylamide gel electrophoresis. The portion of DNA template without protein will be cut at random locations, and thus when it is run on a gel, will produce a ladder-like distribution. The DNA template with the protein will result in ladder distribution with a break in it, the "footprint", where the DNA has been protected from the cleavage agent.

Labeling

The DNA template can be labeled at the 3' or 5' end, depending on the location of the binding site(s). Labels that can be used are:

Radioactivity has been traditionally used to label DNA fragments for footprinting analysis, as the method was originally developed from the Maxam-Gilbert chemical sequencing technique. Radioactive labeling is very sensitive and is optimal for visualizing small amounts of DNA.

Fluorescence is a desirable advancement due to the hazards of using radio-chemicals. However, it has been more difficult to optimize because it is not always sensitive enough to detect the low concentrations of the target DNA strands used in DNA footprinting experiments. Electrophoretic sequencing gels or capillary electrophoresis have been successful in analyzing footprinting of fluorescently tagged fragments.[1]

Cleavage agent

A variety of cleavage agents can be chosen. Ideally a desirable agent is one that is sequence neutral, easy to use, and is easy to control. Unfortunately none available meet all these all of these standards, so an appropriate agent can be chosen, depending on your DNA sequence and ligand of interest. The following cleavage agents are described in detail:

DNase I: a large protein that functions as a double-strand endonuclease. It binds the minor groove of DNA and cleaves the phosphodiester backbone. It is a good cleavage agent for footprinting because its size makes it easily physically hindered. Thus is more likely to have its action blocked by a bound protein on a DNA sequence. In addition, the DNase I enzyme is easily controlled by adding EDTA to stop the reaction. There are however some limitations in using DNase I. The enzyme does not cut DNA randomly; its activity is affected by local DNA structure and sequence and therefore results in an uneven ladder. This can limit the precision of predicting a protein’s binding site on the DNA molecule.[1][2]

Hydroxyl radicals: are created from the Fenton reaction, which involves reducing Fe2+ with H2O2 to form free hydroxyl molecules. These hydroxyl molecules react with the DNA backbone, resulting in a break. Due to their small size, the resulting DNA footprint has high resolution. Unlike DNase I they have no sequence dependence and result in a much more evenly distributed ladder. The negative aspect of using hydroxyl radicals is that they are more time consuming to use, due to a slower reaction and digestion time.[3]

Ultraviolet irradiation: can be used to excite nucleic acids and create photoreactions, which results in damaged bases in the DNA strand. Photoreactions can include: single strand breaks, interactions between or within DNA strands, reactions with solvents, or crosslinks with proteins.

The workflow for this method has an additional step, once both your protected and unprotected DNA have been treated, there is subsequent primer extension of the cleaved products. The extension will terminate upon reaching a damaged base, and thus when the PCR products are run side-by-side on a gel; the protected sample will show an additional band where the DNA was crosslinked with a bound protein.

Advantages of using UV are that it reacts very quickly and can therefore capture interactions that are only momentary. Additionally it can be applied to in vivo experiments, because UV can penetrate cell membranes. A disadvantage is that the gel can be difficult to interpret, as the bound protein does not protect the DNA, it merely alters the photoreactions in the vicinity.[4]

Highly developed Analysis

In vivo footprinting

In vivo footprinting is a technique used to analyze the protein-DNA interactions that are occurring in a cell at a given time point. DNase I can be used as a cleavage agent if the cellular membrane has been permeabilized. However the most common cleavage agent used is UV irradiation because it penetrates the cell membrane without disrupting cell state and can thus capture interactions that are sensitive to cellular changes. Once the DNA has been cleaved or damaged by UV, the cells can be lysed and DNA purified for analysis of a region of interest.

Ligation-mediated PCR is an alternative method to footprint in vivo. Once a cleavage agent has been used on the genomic DNA, resulting in single strand breaks, and the DNA is isolated, a linker is added on to the break points. A region of interest is amplified between the linker and a gene-specific primer, and when run on a polyacrylamide gel, will have a footprint where a protein was bound.[5]

In vivo footprinting combined with immunoprecipitation can be used to assess protein specificity at many locations throughout the genome. The DNA bound to a protein of interest can be immunoprecipitated with an antibody to that protein, and then specific region binding can be assessed using the DNA footprinting technique.[6]

Quantitative footprinting

The DNA footprinting technique can be modified to assess the binding strength of a protein to a region of DNA. Using varying concentrations of the protein for the footprinting experiment, the appearance of the footprint can be observed as the concentrations increase and the proteins binding affinity can then be estimated.[1]

Footprinting coupled with detection by capillary electrophoresis

To adapt the footprinting technique to updated detection methods, the labelled DNA fragments are detected by a capillary electrophoresis device instead of being run on a polyacrylamide gel. If the DNA fragment to be analyzed is produced by polymerase chain reaction (PCR), it's is straightforward to couple a fluorescent molecule such as carboxyfluorescein (FAM) to the primers. This way, the fragments produced by DNaseI digestion will contain FAM, and will be detectable by the capillary electrophoresis machine. Typically, carboxytetramethyl-rhodamine (ROX)-labelled size standards are also added to the mixture of fragments to be analyzed. Binding sites of transcription factors have been successfully identified this way. [7]