Single-molecule analysis of human telomerase monomer

Human telomerase is a ribonucleoprotein that is minimally comprised of protein (hTERT) and RNA (hTR) components. We have applied single-molecule fluorescence two-color coincidence detection to characterize complex formation between fluorophore-labeled components in solution. By systematic labeling and in vitro assembly of hTERT, hTR and telomerase's DNA substrate, we have established that catalytically functional human telomerase comprises a stable hTERT:hTR:substrate interaction in a 1:1:1 absolute stoichiometry.     

Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA

RNA unfolding and folding reactions in physiological conditions can be facilitated by mechanical force one molecule at a time. By using force-measuring optical tweezers, we studied the mechanical unfolding and folding of a hairpin-type pseudoknot in human telomerase RNA in a near-physiological solution, and at room temperature. Discrete two-state folding transitions of the pseudoknot are seen at approximately 10 and approximately 5 piconewtons (pN), with ensemble rate constants of approximately 0.1 sec(-1), by stepwise force-drop experiments. Folding studies of the isolated 5'-hairpin construct suggested that the 5'-hairpin within the pseudoknot forms first, followed by formation of the 3'-stem. Stepwise formation of the pseudoknot structure at low forces are in contrast with the one-step unfolding at high forces of approximately 46 pN, at an average rate of approximately 0.05 sec(-1). In the constant-force folding trajectories at approximately 10 pN and approximately 5 pN, transient formation of nonnative structures were observed, which is direct experimental evidence that folding of both the hairpin and pseudoknot takes complex pathways. Possible nonnative structures and folding pathways are discussed.     

Single-molecule studies of DNA replisome function

Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the leading and lagging strand synthesis of DNA. These complex, yet elegantly organized, molecular machines have been studied extensively by kinetic and structural methods to provide an in-depth understanding of the mechanism of DNA replication. Owing to averaging of observables, unique dynamic information of the biochemical pathways and reactions is concealed in conventional ensemble methods. However, recent advances in the rapidly expanding field of single-molecule analyses to study single biomolecules offer opportunities to probe and understand the dynamic processes involved in large biomolecular complexes such as replisomes. This review will focus on the recent developments in the biochemistry and biophysics of DNA replication employing single-molecule techniques and the insights provided by these methods towards a better understanding of the intricate mechanisms of DNA replication.     

Single-molecule analysis of dynein processivity and stepping behavior

Cytoplasmic dynein, the 1.2 MDa motor driving minus-end-directed motility, has been reported to move processively along microtubules, but its mechanism of motility remains poorly understood. Here, using S. cerevisiae to produce recombinant dynein with a chemically controlled dimerization switch, we show by structural and single-molecule analysis that processivity requires two dynein motor domains but not dynein's tail domain or any associated subunits. Dynein advances most frequently in 8 nm steps, although longer as well as side and backward steps are observed. Individual motor domains show a different stepping pattern, which is best explained by the two motor domains shuffling in an alternating manner between rear and forward positions. Our results suggest that cytoplasmic dynein moves processively through the coordination of its two motor domains, but its variable step size and direction suggest a considerable diffusional component to its step, which differs from Kinesin-1 and is more akin to myosin VI.     

端粒酶的非端粒保护作用

端粒酶与细胞永生化和肿瘤的发生发展密切相关,过去认为是由于端粒酶保护端粒从而阻止因端粒缩短所导致的细胞凋亡;然而近年来,越来越多证据表明:端粒酶在维持端粒长度之外,还存在着非端粒保护作用。通过对其非端粒保护作用的研究,有助于深入而全面地阐明端粒酶的生物学行为及其作用机理,对于肿瘤等疾病的治疗具有重要意义。本文对端粒酶的非端粒保护作用进行小结。     

Single-molecule FRET of protein structure and dynamics - a primer

Single-molecule spectroscopy has developed into a widely used method for probing the structure, dynamics, and mechanisms of biomolecular systems, especially in combination with Förster resonance energy transfer (FRET). In this introductory tutorial, essential concepts and methods will be outlined, from the FRET process and the basic considerations for sample preparation and instrumentation to some key elements of data analysis and photon statistics. Different approaches for obtaining dynamic information over a wide range of timescales will be explained and illustrated with examples, including the quantitative analysis of FRET efficiency histograms, correlation spectroscopy, fluorescence trajectories, and microfluidic mixing.     

Single-molecule and population probing of chromatin structure using DNA methyltransferases

Probing chromatin structure with DNA methyltransferases offers advantages over more commonly used nuclease-based and chromatin immunoprecipitation methods for detection of nucleosomes and non-histone protein-DNA interactions. Here, we describe two related methods in which the readout of MTase accessibility is obtained by assaying 5-methylcytosine in DNA through the PCR-based technique of bisulfite genomic sequencing. The methyltransferase accessibility protocol (MAP) determines the relative frequency at which the enzyme accesses each of its target sites over an entire population of PCR amplified product. While MAP yields much quantitative information about relative accessibility of a region of chromatin, a complementary single-molecule view of methyltransferase accessibility, termed MAP for individual templates (MAP-IT), is provided by analysis of cloned PCR products. Absolute rather than relative methylation frequencies in a region are obtained by summing the methylation status at each site over a cohort of clones. Moreover, as the integrity of individual molecules is maintained in MAP-IT, unique information about the distribution of multiple footprints along continuous regions is gleaned. In principle, the population MAP and single-molecule MAP-IT strategies can be used to analyze chromatin structure in a variety of model systems. Here, we describe the application of MAP in living Saccharomyces cerevisiae cells and MAP-IT in the analysis of a mammalian tumor suppressor gene in nuclei. This application of MAP-IT provides the first means to simultaneously determine CpG methylation of mammalian genes and their overlying chromatin structure in the same single DNA molecule.     

Single-molecule analysis of genome rearrangements in cancer

Rearrangements of the genome can be detected by microarray methods and massively parallel sequencing, which identify copy-number alterations and breakpoint junctions, but these techniques are poorly suited to reconstructing the long-range organization of rearranged chromosomes, for example, to distinguish between translocations and insertions. The single-DNA-molecule technique HAPPY mapping is a method for mapping normal genomes that should be able to analyse genome rearrangements, i.e. deviations from a known genome map, to assemble rearrangements into a long-range map. We applied HAPPY mapping to cancer cell lines to show that it could identify rearrangement of genomic segments, even in the presence of normal copies of the genome. We could distinguish a simple interstitial deletion from a copy-number loss at an inversion junction, and detect a known translocation. We could determine whether junctions detected by sequencing were on the same chromosome, by measuring their linkage to each other, and hence map the rearrangement. Finally, we mapped an uncharacterized reciprocal translocation in the T-47D breast cancer cell line to about 2 kb and hence cloned the translocation junctions. We conclude that HAPPY mapping is a versatile tool for determining the structure of rearrangements in the human genome.     

Single-molecule analysis of DNA-protein complexes using nanopores

We present a method for rapid measurement of DNA-protein interactions using voltage-driven threading of single DNA molecules through a protein nanopore. Electrical force applied to individual ssDNA-exonuclease I complexes pulls the two molecules apart, while ion current probes the dissociation rate of the complex. Nanopore force spectroscopy (NFS) reveals energy barriers affecting complex dissociation. This method can be applied to other nucleic acid-protein complexes, using protein or solid-state nanopore devices.     

Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo

Vault poly(ADP-ribose) polymerase (VPARP) was originally identified as a minor protein component of the vault ribonucleoprotein particle, which may be involved in molecular assembly or subcellular transport. In addition to the association of VPARP with the cytoplasmic vault particle, subpopulations of VPARP localize to the nucleus and the mitotic spindle, indicating that VPARP may have other cellular functions. We found that VPARP was associated with telomerase activity and interacted with exogenously expressed telomerase-associated protein 1 (TEP1) in human cells. To study the possible role of VPARP in telomerase and vault complexes in vivo, mVparp-deficient mice were generated. Mice deficient in mVparp were viable and fertile for up to five generations, with no apparent changes in telomerase activity or telomere length. Vaults purified from mVparp-deficient mouse liver appeared intact, and no defect in association with other vault components was observed. Mice deficient in mTep1, whose disruption alone does not affect telomere function but does affect the stability of vault RNA, showed no additional telomerase or telomere-related phenotypes when the mTep1 deficiency was combined with an mVparp deficiency. These data suggest that murine mTep1 and mVparp, alone or in combination, are dispensable for normal development, telomerase catalysis, telomere length maintenance, and vault structure in vivo.     

Secondary structure of vertebrate telomerase RNA

Telomerase is a ribonucleoprotein enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The essential RNA component of telomerase provides the template for telomeric repeat synthesis. To determine the secondary structure of vertebrate telomerase RNA, 32 new telomerase RNA genes were cloned and sequenced from a variety of vertebrate species including 18 mammals, 2 birds, 1 reptile, 7 amphibians, and 4 fishes. Using phylogenetic comparative analysis, we propose a secondary structure that contains four structural domains conserved in all vertebrates. Ten helical regions of the RNA are universally conserved while other regions vary significantly in length and sequence between different classes of vertebrates. The proposed vertebrate telomerase RNA structure displays a strikingly similar topology to the previously determined ciliate telomerase RNA structure, implying an evolutionary conservation of the global architecture of telomerase RNA.     

Dynamic analysis of the structure and function of sarcomeres

We attempted to analyze the relationships between the steric structure of the sarcomere and its physiological functions by the use of a sarcomere model of muscle contraction, which includes the geometric arrangement of the thick and thin filaments of the sarcomere, as well as of the cross-bridges and actin sites. Motions of both cross-bridges and myofilaments were considered in terms of our three-state model of the elementary cycle under constraints caused by the steric structure of the sarcomere proposed by Huxley and Brown. Each cross-bridge moves in a molecular potential of our three-state model under the influence of the sliding motions of myofilaments. The sarcomere model described well the tension-velocity relation and isotonic transient processes quantitatively and consistently. In addition, it allowed independence of the no-load shortening velocity upon the overlap of the thick and thin filaments, although the motions of cross-bridges were not independent. Effects of the helical periodicities of the thick and thin filaments and of the number of cross-bridges upon muscle contraction were studied, and the conditions for smooth and efficient contraction of muscle were obtained.     

NMR analysis of structure and function of snake neurotoxins

The 270-MHz proton-nmr spectra of short neurotoxins (erabutoxins from Laticauda semifasciata and cobrotoxin from Naja naja atra) and long neurotoxins (toxin B from Naja naja and α-bungarotoxin from Bungarus multicinctus) have been analyzed. The conformation of erabutoxin b in solution is largely consistent with the x-ray crystal analysis, although the environment of His-7 in solution is definitely different from that in the crystal. The pH-dependent transition has been found for toxin B, indicating that the conformation in neutral solution is different from that in the crystal as grown from acidic solution. The deuterium-exchange rates of the amide protons for the four neurotoxins have been measured. The order of structural rigidity is the same as the order of the irreversibility of neuromuscular block by neurotoxins.     

Factor VIII: structure, function and analysis

Factor VIII is an important blood coagulation protein whose genetic deficiency leads to the serious bleeding disorder, classic haemophilia (haemophilia A).Here we review the structure, function and analysis of this protein for diagnostic and therapeutic applications. Because factor VIII is tightly associated with von Willebrand factor some recent work on the latter is also considered so as to clarify the relationship between them.