TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres

Abstract

Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1’s 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼9–17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼2.8–3.6 κBT greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This ‘tag-team proofreading’ represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.

Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process

“…Subdiffusion of macromolecules in biological systems has been observed previously (Dunn et al, 2011; Ghodke et al, 2014; Gorman et al, 2007; Hofling and Franosch, 2013; Hughes et al, 2013; Lin et al, 2014). Rad4 showed increased constrained motion at physiological salt concentrations (Figure 2), which could be due to the faborable hydrophobic interactions between aromatic side-chains (F556, F597, and F599) and DNA bases at elevated ionic strengths.…”

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

“…32, and single-molecule methods have been used to experimentally validate and visualize various protein search strategies (27,28,(33)(34)(35). We have previously developed a DNA tightrope assay that enables the direct visualization of dynamics of QD-conjugated proteins on DNA (27)(28)(29)(30). Briefly, in this assay, λ-DNA tightropes are strung-up between 5-μm poly-L-lysinecoated beads, which are deposited on a PEGylated coverslip ( Fig.…”

“…To better understand damage recognition by UV-DDB, we used a single-molecule DNA tightrope assay (27)(28)(29)(30) to observe the real time interactions of quantum dot (QD)-conjugated wildtype (WT) UV-DDB or UV-DDB containing the K244E mutation in DDB2, with damaged DNA substrates with high temporal and spatial resolution. Observations of individual molecules reveal the presence of short-lived intermediates and heterogeneity in molecular properties that may be lost due to bulk averaging of the properties of an unsynchronized ensemble of molecules.…”

Unraveling secrets of telomeres: One molecule at a time

“…Recently, we used DNA tightrope assay based single-molecule fluorescence imaging to directly probe how TRF1 and TRF2 locate their target DNA sequences and protein partners (Fig. 7) [124] . Fluorescent labeling of TRF1 and TRF2 was achieved by conjugating 6x histidine (His 6 ) tagged TRF1 and TRF2 to streptavidin-conjugated quantum dots (QDs) using the biotinylated multivalent chelator tris-nitrilotriacetic acid ( BT tris-NTA) (Fig.…”

“…DNA tightrope assay based oblique-angle fluorescence imaging of TRF1- and TRF2-QDs on λ DNA tightropes [124]. (A) Schematic representations of TRF1- and TRF2-QD conjugates.…”