Specific anchoring of large topologically closed DNA for single-molecule protein:DNA interactions

Abstract

Single-molecule and bulk biophysical approaches to study protein-DNA interactions on surface-immobilized nucleic acid templates typically rely on modifying the ends of linear DNA molecules to enable surface-DNA attachments. Unless both strands are constrained, this results in topologically free DNA molecules and the inability to observe supercoiling-dependent biological processes or requires additional means to micromanipulate the free DNA end to impose rotational constraints or induce supercoiling. We developed a method using RecA protein to induce the formation of a circularized compliment-stabilized D-loop. The resulting joint molecule is topologically closed, surface anchorable, and stable under microfluidic flow. Importantly, the method obviates the need for subsequent manipulation of surface-tethered DNA; tethered molecules remain supercoiled and retain accessibility to DNA-binding proteins. This approach adds to the toolkit for those studying processes on DNA that require supercoiled DNA templates or topologically constrained systems.

Figure 1

Optimization of RecA-dependent D-loop formation. (A) Schematic of a reaction between a negatively supercoiled plasmid DNA and oligonucleotide with homologous sequence to the plasmid. RecA catalyzes DNA strand exchange between the oligonucleotide and plasmid, inducing the formation of a D-loop structure. (B) Titration of RecA nucleoprotein filaments (10, 20, 50, and 100 nM molecules; RecA:ssDNA = 2:1) against a fixed concentration of plasmid (14.5 nM, molecules). (C) RecA dependency of D-loop formation. Deproteinized D-loops were separated from the free oligonucleotide by native agarose gel electrophoresis.

Figure 2

Production of a topologically closed cs-D-loop. (A) Schematic of a reaction that generates a cs-D-loop with juxtaposed ligatable ends. After RecA induces the formation of a D-loop using an oligonucleotide with sequence homology to the negatively supercoiled plasmid, a second oligonucleotide that is complimentary to the displaced strand can anneal, forming a cs-D-loop that is topologically trapped upon ligation. A gold asterisk indicates the position of the biotin-dT moiety. (B) Verification of cyclization of the cs-D-loop using heat stability as an indirect readout of topological entrapment. Red asterisk indicates a spurious annealing product after heat denaturation (C) Oligonucleotide and RecA dependencies of cs-D-loop formation. The 106-mer oligonucleotide containing the internal biotin modification is radiolabeled and is present in every lane.

Figure 3

Single-molecule imaging of anchored plasmids and their in situ linearization with a restriction enzyme. (A) (Left) Micrograph of anchored plasmids stained with SYTOX Green in the absence of microfluidic flow. (Middle) The same field of view experiencing microfluidic flow. (Right) The same field of view under flow after cleavage with BmtI-HF restriction endonuclease. The scale bar represents a length of 10 μm. (B) Kymograph of an anchored DNA molecule. The black arrow indicates when the molecule was cleaved by BmtI-HF; note the sudden extension of the DNA. Black and red asterisks indicate when flow was turned off and on, respectively. (C) Quantification of molecule lengths in the presence and absence of flow and after linearization, respectively.