In Vitro Methods Used to Study DNA–Protein Interactions

摘要

— The interaction of proteins with DNA underlies all processes of cell functioning. They include DNA transcription, replication, and repair; maintenanc of the chromosome structure, activation and inhibition of the genes by protein transcription factors and protein enzymes involved in chromatin modification; maintenance of the nucleosome structure, the control of gene expression, etc. Transcription factors (TFs) bind to specific sets of DNA sequences, activating or inhibiting gene transcription, and are involved in a variety of signal transmission processes, including changes in cell differentiation, development, and environmental influences. This important role of DNA interactions with transcription factors led to their active study for many years and to the development of different methods to determine binding sites on the DNA matrix and the proteins interacting with them, including footprinting, Southern and Western blot assays, the systematic evolution of ligands via exponential enrichment (SELEX), chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), surface plasmon resonance (SPR), etc. Each method has advantages and disadvantages. The methods and approaches used to study TF/DNA binding can be divided into in vivo and in vitro. In vivo methods (e.g., ChIP) are used to study the TF/DNA interactions happening under specific conditions, including the tissue type, cells, moment of time, etc. and to scan TF binding sites throughout the genome. In vitro methods are used to study the TF/DNA interaction caused only by the protein and DNA structure to determine the binding site sequences, interaction forces, and characteristics of TF/DNA complexes. The review will list the most widely used in vitro methods for the study of DNA–protein interactions, since there are good reviews for each of them. The footprinting method and the methods to determine the DNA/protein affinity, binding rates, and dissociation of DNA/protein complexes will be discussed in detail.