ABSTRACT
We employed infrared scattering-type scanning near-field optical microscopy (IR-sSNOM) to study surface plasmon polaritons (SPPs) in trilayer graphene (TLG). Our study reveals systematic differences in near-field IR spectra and SPP wavelengths between Bernal (ABA) and rhombohedral (ABC) TLG domains on SiO2, which can be explained by stacking-dependent intraband conductivities. We also observed that the SPP reflection profiles at ABA-ABC boundaries could be mostly accounted for by an idealized domain boundary defined by the conductivity discontinuity. However, we identified distinct shapes in the SPP profiles at the edges of the ABA and ABC TLG, which cannot be solely attributed to idealized edges with stacking-dependent conductivities. Instead, this can be explained by the presence of various edge structures with local conductivities differing from those of bulk TLGs. Our findings unveil a new structural element that can control SPP, and provide insights into the structures and electronic states of the edges of few-layer graphene.
ABSTRACT
Topoisomerase II cleaves DNA at preferred sequences with different efficiencies; however, the mechanism of cleavage site selection is not known. Here we used single-molecule fluorescence assays that monitor several critical steps of DNA-topoisomerase II interactions, including binding/dissociation, bending/straightening, and cleavage/religation, and reveal that the cleavage site is selected mainly during the bending step. Furthermore, despite the sensitivity of the bending rate to the DNA sequence, it is not an intrinsic property of the DNA itself. Rather, it is determined by protein-DNA interactions.
Subject(s)
DNA Cleavage , DNA Topoisomerases, Type II/metabolism , DNA, Fungal/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Base Sequence , DNA, Fungal/chemistry , Nucleic Acid Conformation , Protein Binding , Saccharomyces cerevisiae/chemistry , Substrate SpecificityABSTRACT
Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA-guided DNA endonuclease, which provides an alternative to type II CRISPR-Cas9 for genome editing. Previous studies have revealed that Cas12a has unique features distinct from Cas9, but the detailed mechanisms of target searching and DNA cleavage by Cas12a are still unclear. Here, we directly observe this entire process by using single-molecule fluorescence assays to study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that AsCas12a ribonucleoproteins search for their on-target site by a one-dimensional diffusion along elongated DNA molecules and induce cleavage in the two DNA strands in a well-defined order, beginning with the non-target strand. Furthermore, the protospacer-adjacent motif (PAM) for AsCas12a makes only a limited contribution of DNA unwinding during R-loop formation and shows a negligible role in the process of DNA cleavage, in contrast to the Cas9 PAM.
Subject(s)
Acidaminococcus/genetics , CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , DNA/genetics , RNA, Guide, Kinetoplastida/genetics , Acidaminococcus/enzymology , Base Pairing , Base Sequence , CRISPR-Associated Protein 9/metabolism , Cloning, Molecular , DNA/metabolism , DNA Cleavage , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Editing , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Models, Molecular , Nucleic Acid Conformation , Nucleic Acid Heteroduplexes , RNA, Guide, Kinetoplastida/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Homotypic fusion of endoplasmic reticulum membranes is driven by atlastin GTPases; however, the underlying mechanism remains largely unknown. Here, using a FRET-based single-vesicle fusion assay with liposomes bearing the yeast atlastin Sey1p, we investigated the molecular mechanisms of atlastin-mediated membrane tethering and fusion. Although Sey1p-bearing proteoliposomes frequently underwent membrane tethering in a GTP hydrolysis-dependent manner as reported in studies using bulk assays, only a small fraction of the tethered liposomes proceeded to fusion. Strikingly, the rest of the tethered liposomes failed to fuse or dissociate. This stable tethering, however, did not require continued GTP hydrolysis because GTP omission and magnesium chelation did not disrupt tethering. Interestingly, an increased Sey1p density on the membrane markedly accelerated tethering but barely affected the fusion rate of the tethered liposomes, indicating that Sey1p requires additional factors to support efficient fusion in vivo. Finally, the assay also revealed that Sey1p-mediated liposome fusion occurs through hemifusion, suggesting the mechanistic conservation between biological membrane fusion events despite the existence of diverse fusogens.
