Assembly of DNA Curtains Using Hydrogen Silsesquioxane As a Barrier to Lipid Diffusion
ABSTRACT We have established a single-molecule imaging experimental platform called "DNA curtains" in which DNA molecules tethered to a lipid bilayer are organized into patterns at nanofabricated metallic barriers on the surface of a microfluidic sample chamber. This technology has wide applications for real-time single-molecule imaging of protein-nucleic acid interactions. Here, we demonstrate that DNA curtains can also be made from hydrogen silsesquioxane (HSQ). HSQ offers important advantages over metallic barriers because it can be lithographically patterned directly onto fused silica slides without any requirement for further processing steps, thereby offering the potential for rapid prototype development and/or scale up for manufacturing.
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ABSTRACT: In physiological settings, nucleic-acid translocases must act on substrates occupied by other proteins, and an increasingly appreciated role of translocases is to catalyse protein displacement from RNA and DNA. However, little is known regarding the inevitable collisions that must occur, and the fate of protein obstacles and the mechanisms by which they are evicted from DNA remain unexplored. Here we sought to establish the mechanistic basis for protein displacement from DNA using RecBCD as a model system. Using nanofabricated curtains of DNA and multicolour single-molecule microscopy, we visualized collisions between a model translocase and different DNA-bound proteins in real time. We show that the DNA translocase RecBCD can disrupt core RNA polymerase, holoenzymes, stalled elongation complexes and transcribing RNA polymerases in either head-to-head or head-to-tail orientations, as well as EcoRI(E111Q), lac repressor and even nucleosomes. RecBCD did not pause during collisions and often pushed proteins thousands of base pairs before evicting them from DNA. We conclude that RecBCD overwhelms obstacles through direct transduction of chemomechanical force with no need for specific protein-protein interactions, and that proteins can be removed from DNA through active disruption mechanisms that act on a transition state intermediate as they are pushed from one nonspecific site to the next.Nature 12/2010; 468(7326):983-7. DOI:10.1038/nature09561 · 42.35 Impact Factor
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ABSTRACT: We have used total internal reflection fluorescence microscopy (TIRFM) to investigate the characteristics of the yeast homologous recombination factor Rdh54 on DNA. Our results demonstrate translocation of Rdh54 on DNA and extrusion of DNA loops by Rdh54 in an ATP hydrolysis-dependent manner. The translocating Rdh54 was highly processive and displayed a variety of behavior, including variations in translocation rate and distance, pauses, and reversals. We provide evidence that the DNA loops generated encompass an average of 6 kb, and Rdh54 often abruptly releases the extruded DNA. Rdh54 forms a multimeric complex, which we speculate has at least two functionally distinct DNA-binding sites, one of which enables translocation while the other remains anchored to another DNA locale. Our work, together with other recent studies, suggests that translocation-coupled DNA loop extrusion is a common mechanistic feature among the Snf2-family of chromatin-remodeling proteins.Journal of Molecular Biology 07/2007; 369(4):940-53. DOI:10.1016/j.jmb.2007.04.005 · 3.96 Impact Factor
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ABSTRACT: Here we use single-molecule imaging to determine coarse-grained intrinsic energy landscapes for nucleosome deposition on model DNA substrates. Our results reveal distributions that are correlated with recent in silico predictions, reinforcing the hypothesis that DNA contains some intrinsic positioning information. We also show that cis-regulatory sequences in human DNA coincide with peaks in the intrinsic landscape, whereas valleys correspond to nonregulatory regions, and we present evidence arguing that nucleosome deposition in vertebrates is influenced by factors that are not accounted for by current theory. Finally, we demonstrate that intrinsic landscapes of nucleosomes containing the centromere-specific variant CenH3 are correlated with patterns observed for canonical nucleosomes, arguing that CenH3 does not alter sequence preferences of centromeric nucleosomes. However, the nonhistone protein Scm3 alters the intrinsic landscape of CenH3-containing nucleosomes, enabling them to overcome the otherwise exclusionary effects of poly(dA-dT) tracts, which are enriched in centromeric DNA.Nature Structural & Molecular Biology 10/2009; 16(10):1056-62. DOI:10.1038/nsmb.1655 · 11.63 Impact Factor