Multimerization of Restriction Fragments by Magnesium-Mediated Stable Base Pairing between Overhangs:  A Cause of Electrophoretic Mobility Shift

Abstract

The electrophoretic mobility shift assay (EMSA) or simply the "gel shift assay" is one of the most sensitive methods for studying the ability of a protein to bind to DNA. EMSAs are also widely used to investigate protein- or sequence-dependent DNA bending. Here we report that electrophoresis using physiological concentrations of Mg(2+) can cause a mobility shift of restriction fragments in nondenaturing polyacrylamide gels as the overhangs form stable base pairs. This phenomenon was observed at even 37 degrees C. The retardation was, however, more pronounced at low temperatures, where a three-nucleotide overhang 5'-GAC also caused a mobility shift. The stability of the pairing was generally high when the overhangs of four nucleotides display high GC content, while the mobility shift caused by 5'-AATT was greater than those caused by 5'-GATC, 5'-TCGA, and 5'-CTAG. This observation should be taken into account to avoid misinterpretation of the data when the EMSA, especially the circular permutation gel mobility shift assay, is performed using a running buffer that contains Mg(2+) ions. The stable adhesion between short overhangs may present an important basis for genome stability and many genetic processes occurring in living cells.

Self-Assembly of Double-Stranded DNA Molecules at Nanomolar Concentrations

“…The assembled molecules would migrate more slowly than the nonassembled constituents. In an experiment using DNA fragments with two cohesive ends, we previously observed more pronounced band shifts or smearing (12), while in the study, we did not observe retarded migration of the fragments with blunt ends. On the basis of these observations, we argued that cohesive ends form stable base pairings that resist electrophoretic separation.…”

“…The electrophoretic field across the gels was maintained at 10.8 V/cm. The buffer solution was pumped between the electrode compartments as described previously (12). DNA Ligation.…”

“…We have previously reported that electrophoresis using physiological concentrations of Mg 2+ can cause a mobility shift of restriction fragments in nondenaturing polyacrylamide gels as the overhangs interact (12). Theoretically, this phenomenon can be used to detect preferential intermolecular DNA interactions, if any, in aqueous solutions.…”

Phenolic Acid-Mediated Regulation of the padC Gene, Encoding the Phenolic Acid Decarboxylase of Bacillus subtilis

“…Thus, it is very often used in mobility shift assay buffers to facilitate binding. However, MgCl 2 can induce misleading gel shift in EMSA because of its ability to enhance end-to-end DNA interactions and stable base pairing between overhangs as short as 3 bp (33). Taking into account this potential effect of MgCl 2 in the regulation of intramolecular interactions, we hypothesized that MgCl 2 could counteract the effect of phenolic acids by making the structure of PadR less accessible to phenolic acids and/or by enhancing DNA-protein binding at a level not encountered in living BS168 cells.…”

Quantitative analysis of EcoR1 methylase‐DNA complex by atomic force microscopy

“…DNA-protein interactions play a crucial role in the regulation of normal cellular function. The electrophoretic mobility shift assay (EMSA) (Tagashira et al 2002) and DNA footprinting (Fechter and Dervan 2003) are commonly used for obtaining preliminary information about the local geometry and putative sites of contact within a DNAprotein complex. Unfortunately, both these methods only give limited information on the structures of short DNA fragments.…”