Single-Molecule Tethered Particle Motion: Stepwise Analyses of Site-Specific DNA Recombination

Abstract

Tethered particle motion/microscopy (TPM) is a biophysical tool used to analyze changes in the effective length of a polymer, tethered at one end, under changing conditions. The tether length is measured indirectly by recording the Brownian motion amplitude of a bead attached to the other end. In the biological realm, DNA, whose interactions with proteins are often accompanied by apparent or real changes in length, has almost exclusively been the subject of TPM studies. TPM has been employed to study DNA bending, looping and wrapping, DNA compaction, high-order DNA–protein assembly, and protein translocation along DNA. Our TPM analyses have focused on tyrosine and serine site-specific recombinases. Their pre-chemical interactions with DNA cause reversible changes in DNA length, detectable by TPM. The chemical steps of recombination, depending on the substrate and the type of recombinase, may result in a permanent length change. Single molecule TPM time traces provide thermodynamic and kinetic information on each step of the recombination pathway. They reveal how mechanistically related recombinases may differ in their early commitment to recombination, reversibility of individual steps, and in the rate-limiting step of the reaction. They shed light on the pre-chemical roles of catalytic residues, and on the mechanisms by which accessory proteins regulate recombination directionality.

A bipartite thermodynamic-kinetic contribution by an activating mutation to RDF-independent excision by a phage serine integrase

“…In TPM analysis, the individual steps of recombination from free substrate → protein bound intermediate complexes → product(s) is reported by finite differences in the BM amplitudes of a polystyrene bead attached to one end of the linear substrate DNA molecule whose other end is tethered to a glass slide ( 29 , 34 , 37–38 ). An unbound substrate molecule has a ‘high’ BM amplitude, one occupied by Int dimers at the att sites present within it has an ‘intermediate’ BM amplitude, and one in which the bound att sites have synapsed by looping of the intervening DNA has a ‘low’ BM amplitude (Figure 2 and Supplementary Figure S1 ).…”

“…Single molecule analysis of site-specific DNA recombination and DNA transposition have raised the mechanistic analysis of these reactions to a level of resolution that is not attainable by standard ensemble biochemical studies ( 37–38 , 42 , 44–48 ). The analytical tools employed include magnetic tweezers, TPM, TFM (tethered fluorophore motion), TFM-FRET (fluorescence resonance energy transfer) and PIFE (protein induced fluorescence enhancement).…”

An Ultra-Stable and Dense Single-Molecule Click Platform for Sensing Protein-DNA Interactions

“…straightening, looping or kinking) of the DNA tether. Utilizing TPM, protein-DNA interaction, 6,7 transcription 3,8 and DNA looping 9,10 have been successfully studied in vitro . Furthermore, significant effort has gone into developing TPM derived sensing techniques to monitor the presence and concentration of medically relevant molecules over extended periods of time, making TPM a promising new method for biosensing applications.…”

An Ultrastable and Dense Single‐Molecule Click Platform for Sensing Protein–Deoxyribonucleic Acid Interactions

“…[ 4,5 ] The Brownian motion reflects changes in the flexibility or conformation (e.g., straightening, looping or kinking) of the DNA tether. Utilizing TPM, protein–DNA interaction, [ 6,7 ] transcription, [ 3,8 ] and DNA looping [ 9,10 ] have been successfully studied in vitro. Furthermore, significant effort has gone into developing TPM‐derived sensing techniques to monitor the presence and concentration of medically relevant molecules over extended periods of time, making TPM a promising new method for biosensing applications.…”