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.
Highlights
Tethered particle motion (TPM) analysis is a relatively simple and affordable biophysical method for following the dynamics of long polymers in solution, and changes in these dynamics with changes in environment or as a result of their interaction with ligands
We have taken advantage of TPM to understand the mechanism of action of site-specific recombinases—initially the tyrosine recombinases, Cre and Flp, and subsequently the serine recombinase, φC31 integrase
In conjunction with relatively low or large force fields induced by flow or applied using optical/magnetic tweezers, respectively, TPM can be adapted to address specialized areas of DNA–protein interactions—protein mediated compaction or flexibility changes in DNA induced by architectural proteins, bridging of
Summary
Tethered particle motion (TPM) analysis is a relatively simple and affordable biophysical method for following the dynamics of long polymers in solution, and changes in these dynamics with changes in environment or as a result of their interaction with ligands. TPM has been most commonly used to characterize the interactions of enzyme and non-enzyme proteins with nucleic acids—almost exclusively DNA [1,2,3]. A rare example of the application of TPM to RNA is in characterizing the changes in conformational dynamics and compaction induced by Mg++ ions [4]. The purpose of this review is to provide a brief general introduction to TPM, followed by a summary of its application towards gaining mechanistic insights into the action of two distinct classes of DNA site-specific recombinases
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