Biochemistry


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0 Q&A 2668 Views Jul 20, 2021

Protein translocation on DNA represents the key biochemical activity of ssDNA translocases (aka helicases) and dsDNA translocases such as chromatin remodelers. Translocation depends on DNA binding but is a distinct process as it typically involves multiple DNA binding states, which are usually dependent on nucleotide binding/hydrolysis and are characterized by different affinities for the DNA. Several translocation assays have been described to distinguish between these two modes of action, simple binding as opposed to directional movement on dsDNA. Perhaps the most widely used is the triplex-forming oligonucleotide displacement assay. Traditionally, this assay relies on the formation of a DNA triplex from a dsDNA segment and a short radioactively labeled oligonucleotide. Upon translocation of the protein of interest along the DNA substrate, the third DNA strand is destabilized and eventually released off the DNA duplex. This process can be visualized and quantitated by polyacrylamide electrophoresis. Here, we present an effective, sensitive, and convenient variation of this assay that utilizes a fluorescently labeled oligonucleotide, eliminating the need to radioactively label DNA. In short, our protocol provides a safe and user-friendly alternative.


Graphical abstract:



Figure 1. Schematic of the triplex-forming oligonucleotide displacement assay.


0 Q&A 2024 Views Dec 5, 2020

DNA footprinting is a classic technique to investigate protein-DNA interactions. However, traditional footprinting protocols can be unsuccessful or difficult to interpret if the binding of the protein to the DNA is weak, the protein has a fast off-rate, or if several different protein-DNA complexes are formed. Our protocol differs from traditional footprinting protocols, because it provides a method to isolate the protein-DNA complex from a native gel after treatment with the footprinting agent, thus removing the bound DNA from the free DNA or other protein-DNA complexes. The DNA is then extracted from the isolated complex before electrophoresis on a sequencing gel to determine the footprinting pattern. This analysis provides a possible solution for those who have been unable to use traditional footprinting methods to determine protein-DNA contacts.

0 Q&A 9002 Views Apr 20, 2015
Protein-DNA interaction is a very important cellular process, by which regulation of DNA biological function, usually gene expression, is exerted. The method of random DNA binding selection assay (RDSA) can be used to identify DNA elements bound by proteins with DNA-binding activities. This method is based on the enrichment of the target DNA element by the immobilized recombinant protein on special beads and repeated PCR amplification.
0 Q&A 12337 Views Feb 20, 2015
This protocol describes a robust technique for the measurement of ZMYND11-DNA interaction by a label-free Biolayer Interferometry (BLI). ZMYND11 is a novel histone reader protein that specifically recognizes H3.3K36me3 via its tandem Bromodomain, zinc-finger and PWWP domain (BP). ZMYND11 links the histone-variant-mediated transcription elongation control to tumour suppression and may therefore represent a novel class of drug targets. Like other PWWP domains, ZMYND11 PWWP domain shows highly positively charged surface and interacts with DNA. Previously reported methods include NMR, FP or EMSA. Biolayer interferometry (BLI) is an emerging technology for analyzing all kinds of biomolecular interactions, such as protein-protein and protein-DNA binding. BLI allows for the real time monitoring of the interactions between biomolecules without the need for reagents with enzymatic, fluorescent, or radioactive labels. The technology is based upon the changes in interference pattern of light reflected from the surface of an optical fiber when materials bind to the tip of the fiber. The technique represents an alternative to technologies such as surface plasmon resonance, providing a simple platform that enables label-free monitoring of biomolecular interactions without the use of flow cells. Label-free biosensor methods provide information on binding, kinetics, concentration, and the affinity of an interaction.



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