Interaction studies

DNA-Protein interactions:

DNA-Protein interactions play an important role in any living cell. It controls cellular processes such as replication, transcription, recombination, DNA repair. There are many types of proteins found in a cell. Only those proteins interact with DNA, which have the DNA binding domains. These DNA binding domains possess an affinity to bind to double stranded or single stranded DNA. There are mainly two types of DNA-Protein interactions: 

1) Sequence specific DNA binding: 

In case of sequence specific DNA protein interactions, a DNA binding protein binds to a DNA on a site having a specific nucleotide sequence. 

2) Sequence non-specific DNA binding

In case of sequence non-specific DNA protein interactions, the DNA binding protein can bind to a DNA in a random position on the DNA.

Techniques used for DNA-Protein Interaction:

1) Electrophoretic Mobility Shift Assay:

This technique is based on the observation that Protein–DNA complexes are heavier and migrate more slowly than free linear DNA fragments when subjected to non-denaturing polyacrylamide or agarose gel electrophoresis.

The rate of DNA migration is shifted or retarded when bound to protein, the assay is also referred to as a gel shift or gel retardation assay.

Advantages:

EMSA is a relatively simple in vitro technique to study DNA–protein interactions.

EMSA is its ability to resolve complexes of different stoichiometry or conformation.

Another advantage the source of the DNA-binding protein may be crude nuclear or whole cell extract, in vitro transcription product or a purified preparation.

Disadvantages:

The possibility that the electrophoretic mobility of the nucleotide-protein complex is not only influenced by its size, and the inability of electrophoretic mobility shift assay to detect specific nucleotide sequences to which the protein binds.

2) Foot printing assay:

This technique is used to decode the specific sequence to which a DNA-binding protein or molecule binds. The procedure employs chemical or enzymatic digestion of naked- and protein bound-DNA oligomers. Both the reactions are then compared using gel electrophoresis.

DNaseI Foot Printing is the most commonly used foot printing assay. DNAase I is a double-strand-specific endonuclease, which binds to the minor groove to break phosphodiester bonds.

Limits:

It does not provide identity of the protein. Because of the large molecular weight of DNAse I, its attack is easily sterically hindered, by the bound protein.

3) Southwestern assay:

This technique combines the principles of southern and western blotting and is primarily used for explaining the molecular weight of the protein in a protein–DNA complex.

The crude or purified cytoplasmic, nucleic or whole cell extract containing the protein of interest, is resolved on an SDS-PAGE, followed by electrophoretic transfer of the proteins from the gel to a membrane under conditions favoring renaturation of the proteins. The membrane-bound proteins are then incubated with oligonucleotides to which the protein of interest putatively binds. The membrane is developed, photographed and only the band corresponding to the bound oligo appears in the final picture.

Disadvantage:

DNA-binding proteins involving multiple subunits may get dissociated during the SDS-PAGE step and hence evade detection. Even the proteins which are monomers may not renature properly on the blot to recognize their binding sequence.

4) Yeast one-hybrid assay:

It is genetic method for identifying proteins that can interact with a DNA sequence of interest. The assay is similar to the widely used yeast two-hybrid assay that identifies protein-protein interactions, either in small or large-scale settings.

Advantage:

The yeast one-hybrid technique could help analyze DNA–protein interactions through the expression of reporter genes. 

The advantage of cloning transcription factors or other DNA-binding proteins via one-hybrid screenings, compared to biochemical techniques, is that the procedure does not require specific optimization of in vitro conditions.

Disadvantage:

The problems of false positives and false negatives are the major disadvantages of this technique. False positives are usually caused by high recognition of the bait sequence by endogenous yeast TFs so that the reporter gene could be activated in the absence of prey.

5) Chromatin immunoprecipitation (ChIP):

Chromatin immunoprecipitation has proven to be an excellent experimental method used to determine the in vivo analysis of DNA–protein interactions.

The methodology of chip involves shearing of protein associated chromatin into smaller fragments followed by immunoprecipitating the DNA–protein complex using protein-specific antibody.

The isolated DNA–protein complexes are then dissociated and the specifically enriched DNA segment is analyzed using PCR amplification methods.

Protein-Protein interactions:

Proteins control all biological systems in a cell, and while many proteins perform their functions independently, the vast majority of proteins interact with others for proper biological activity.

1) Tandem affinity purification-mass spectroscopy (TAP-MS):

TAP-MS is based on the double tagging of the protein of interest on its chromosomal locus, followed by a two-step purification process and mass spectroscopic analysis.

Advantage:

There can be real determination of protein partners quantitatively in vivo without prior knowledge of complex composition.

Its simplicity, high yield, and wide applicability make it a very useful procedure for protein purification and proteome exploration.

2) Protein microarrays (H):

Microarray-based analysis allows the simultaneous analysis of thousands of parameters within a single experiment.

It is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. 

Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. 

It only allows the analysis of proteins that have already been discovered.

3) Yeast 2 hybrid (Y2H):

Yeast two-hybrid is typically carried out by screening a protein of interest against a random library of potential protein partners.

The advantages of a Y2H screen include: 

that it is relatively fast and easy way to screen for protein-protein interactions; 

it requires little hands-on time and technical skill and; 

it is also able to be scaled up by screening yeast libraries of tagged “prey” proteins against a single “bait”, allowing

Limits:

One limitation of classic yeast two-hybrid screens is that they are limited to soluble proteins.

Reference: 

V. Srinivasa Rao, K. Srinivas, G. N. Sujini, G. N. Sunand Kumar, "Protein-Protein Interaction Detection: Methods and Analysis", International Journal of Proteomics, vol. 2014, Article ID 147648, 12 pages, 2014. https://doi.org/10.1155/2014/147648

Flora Cozzolino, Ilaria Iacobucci, Vittoria Monaco, and Maria Monti Journal of Proteome Research 2021 20 (6), 3018-3030 DOI: 10.1021/acs.jproteome.1c0007

Dey, B., Thukral, S., Krishnan, S. et al. DNA–protein interactions: methods for detection and analysis. Mol Cell Biochem 365, 279–299 (2012). https://doi.org/10.1007/s11010-012-1269-z