Cohesin is a chromosome-bound, multisubunit adenosine triphosphatase complex. After loading onto chromosomes, it generates loops to regulate chromosome functions. It has been suggested that cohesin ...organizes the genome through loop extrusion, but direct evidence is lacking. Here, we used single-molecule imaging to show that the recombinant human cohesin-NIPBL complex compacts both naked and nucleosome-bound DNA by extruding DNA loops. DNA compaction by cohesin requires adenosine triphosphate (ATP) hydrolysis and is force sensitive. This compaction is processive over tens of kilobases at an average rate of 0.5 kilobases per second. Compaction of double-tethered DNA suggests that a cohesin dimer extrudes DNA loops bidirectionally. Our results establish cohesin-NIPBL as an ATP-driven molecular machine capable of loop extrusion.
The spatial organization of the genome is critical for fundamental biological processes, including transcription, genome replication, and segregation. Chromatin is compacted and organized with ...defined patterns and proper dynamics during the cell cycle. Aided by direct visualization and indirect genome reconstruction tools, recent discoveries have advanced our understanding of how interphase chromatin is dynamically folded at the molecular level. Here, we review the current understanding of interphase genome organization with a focus on the major regulator of genome structure, the cohesin complex. We further discuss how cohesin harnesses the energy of ATP hydrolysis to shape the genome by extruding chromatin loops.
CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas ...systems, efficient “primed” spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.
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•Cascade diffuses on DNA and transiently engages potential targets•Cascade/Cas3 stalling at protein roadblocks results in DNA double strand breaks•Cascade, Cas3, and Cas1-Cas2 form the primed acquisition complex (PAC)•The PAC displaces protein roadblocks in search of downstream protospacers
Single-molecule studies illuminate how the type I-E CRISPR-Cas interference and adaptation complexes interact and function to achieve primed spacer acquisition.
DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target ...recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2-Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2-Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2-Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2-Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2-Msh3 and Msh2-Msh6 navigate on a crowded genome and suggest how Msh2-Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin.
We present a novel approach for single DNA molecule analysis using neutravidin coated surfaces. DNA molecules are elongated and reversibly immobilized on neutravidin coated surfaces with pH and salt ...controls. We demonstrate restriction enzyme reactions for optical mapping and ligation for tethered DNA molecules.
Single-molecule studies of protein-nucleic acid interactions frequently require site-specific modification of long DNA substrates. The bacteriophage λ is a convenient source of high quality long ...(48.5 kb) DNA. However, introducing specific sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multi-step ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into the λ-phage genome with 90-100% yield. Extrahelical structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of S. cerevisiae Proliferating Cell Nuclear Antigen (yPCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that S. cerevisiae Replication Factor C (yRFC) can load yPCNA onto 5'-ssDNA flaps, (CAG)
triplet repeats, and homoduplex DNA. However, yPCNA remains trapped on the (CAG)
structure, confirming a proposed mechanism for triplet repeat expansion. We anticipate that this molecular toolbox will be broadly useful for other studies that require site-specific modification of long DNA substrates.
In the human cell nucleus, dynamically organized chromatin is the substrate for gene regulation, DNA replication, and repair. A central mechanism of DNA loop formation is an ATPase motor ...cohesin-mediated loop extrusion. The cohesin complexes load and unload onto the chromosome under the control of other regulators that physically interact and affect motor activity. Regulation of the dynamic loading cycle of cohesin influences not only the chromatin structure but also genome-associated human disorders and aging. This review focuses on the recently spotlighted genome organizing factors and the mechanism by which their dynamic interactions shape the genome architecture in interphase.
Although CO
2
emissions are purely global externalities and unlikely to be addressed by individual countries, empirical attention has typically been on the national level as national efforts have ...come in to plug the gap. The contribution of this study is therefore to investigate the effect of population aging on CO
2
emissions, controlling for income and fossil fuel energy consumption, using panel data of 25 OECD countries during 1980–2015. After applying the panel cointegration approach such as fully modified ordinary least squares (FMOLS) and dynamic ordinary least squares (DOLS), we discover that population aging appears to reduce CO
2
emissions, ceteris paribus. We also confirm the existence of the environmental Kuznets curve (EKC) relationship with an inverted-U curve, where CO
2
emissions increase with income level until it reaches the estimated turning point and then starts to turn down. Other evidence shows that fossil fuel energy consumption increases CO
2
emissions, other things equal. We believe that these findings present sustainable policy directions that may help solve the problem of population aging our world is dealing with today.
If the wave mode used in guided wave non-destructive inspection is dispersive, reflected pulses from damaged parts may be significantly distorted due to wave dispersion. The main concern, in this ...case, is how to detect the reflected pulses in noisy signals, and to extract meaningful damage information from the detected pulses. However, current signal processing techniques used for guided wave inspection do not account for pulse dispersion, so the extracted information is often not so accurate. The objective of this study is to develop an efficient technique to deal with dispersed pulses for guided-wave nondestructive evaluation. Our idea is to model dispersed pulses by chirp functions of special form that can simulate up to quadratically varying group delay. To determine the parameters of the chirp functions approximating dispersed, reflected pulses, an adaptive matching pursuit algorithm is employed. Once the characterizing parameters are found, the damage location and extent can be estimated. The proposed method is tested with experimentally measured signals of longitudinal waves in a circular cylinder.
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