Single-Molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA.

Telomeres are essential chromosome end capping structures that safeguard the genome from dangerous DNA processing events. DNA strand invasion occurs during vital transactions at telomeres, including telomere length maintenance by the alternative lengthening of telomeres (ALT) pathway. During telomeric strand invasion, a single-stranded guanine-rich (G-rich) DNA invades at a complementary duplex telomere repeat sequence, forming a displacement loop (D-loop) in which the displaced DNA consists of the same G-rich sequence as the invading single-stranded DNA. Single-stranded G-rich telomeric DNA readily folds into stable, compact, structures called G-quadruplexes (GQs) in vitro and is anticipated to form within the context of a D-loop; however, evidence supporting this hypothesis is lacking. Here, we report a magnetic tweezers assay that permits the controlled formation of telomeric D-loops (TDLs) within uninterrupted duplex human telomere DNA molecules of physiologically relevant lengths. Our results are consistent with a model wherein the displaced single-stranded DNA of a TDL fold into a GQ. This study provides new insight into telomere structure and establishes a framework for the development of novel therapeutics designed to target GQs at telomeres in cancer cells.

Telomere DNA G-quadruplex folding within actively extending human telomerase.

Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K+ versus GQ-destabilizing Li+ salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase-DNA complex. Addition of the telomerase processivity factor POT1-TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li+ cations. This result suggests GQ folding synergizes with POT1-TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K+ but not Li+, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.

Single-molecule FRET-Rosetta reveals RNA structural rearrangements during human telomerase catalysis.

Maintenance of telomeres by telomerase permits continuous proliferation of rapidly dividing cells, including the majority of human cancers. Despite its direct biomedical significance, the architecture of the human telomerase complex remains unknown. Generating homogeneous telomerase samples has presented a significant barrier to developing improved structural models. Here we pair single-molecule Förster resonance energy transfer (smFRET) measurements with Rosetta modeling to map the conformations of the essential telomerase RNA core domain within the active ribonucleoprotein. FRET-guided modeling places the essential pseudoknot fold distal to the active site on a protein surface comprising the C-terminal element, a domain that shares structural homology with canonical polymerase thumb domains. An independently solved medium-resolution structure of Tetrahymena telomerase provides a blind test of our modeling methodology and sheds light on the structural homology of this domain across diverse organisms. Our smFRET-Rosetta models reveal nanometer-scale rearrangements within the RNA core domain during catalysis. Taken together, our FRET data and pseudoatomic molecular models permit us to propose a possible mechanism for how RNA core domain rearrangement is coupled to template hybrid elongation.

Optofluidic wavelength division multiplexing for single-virus detection.

Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context--the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine.

Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid.

Many enzymes promote structural changes in their nucleic acid substrates via application of piconewton forces over nanometer length scales. Magnetic tweezers (MT) is a single molecule force spectroscopy method widely used for studying the energetics of such mechanical processes. MT permits stable application of a wide range of forces and torques over long time scales with nanometer spatial resolution. However, in any force spectroscopy experiment, the ability to monitor structural changes in nucleic acids with nanometer sensitivity requires the system of interest to be held under high degrees of tension to improve signal to noise. This limitation prohibits measurement of structural changes within nucleic acids under physiologically relevant conditions of low stretching forces. To overcome this challenge, researchers have integrated a spatially sensitive fluorescence spectroscopy method, single molecule-FRET, with MT to allow simultaneous observation and manipulation of nanoscale structural transitions over a wide range of forces. Here, we describe a method for using this hybrid instrument to analyze the mechanical properties of nucleic acids. We expect that this method for analysis of nucleic acid structure will be easily adapted for experiments aiming to interrogate the mechanical responses of other biological macromolecules.

The telomerase essential N-terminal domain promotes DNA synthesis by stabilizing short RNA-DNA hybrids.

Telomerase is an enzyme that adds repetitive DNA sequences to the ends of chromosomes and consists of two main subunits: the telomerase reverse transcriptase (TERT) protein and an associated telomerase RNA (TER). The telomerase essential N-terminal (TEN) domain is a conserved region of TERT proposed to mediate DNA substrate interactions. Here, we have employed single molecule telomerase binding assays to investigate the function of the TEN domain. Our results reveal telomeric DNA substrates bound to telomerase exhibit a dynamic equilibrium between two states: a docked conformation and an alternative conformation. The relative stabilities of the docked and alternative states correlate with the number of basepairs that can be formed between the DNA substrate and the RNA template, with more basepairing favoring the docked state. The docked state is further buttressed by the TEN domain and mutations within the TEN domain substantially alter the DNA substrate structural equilibrium. We propose a model in which the TEN domain stabilizes short RNA-DNA duplexes in the active site of the enzyme, promoting the docked state to augment telomerase processivity.

Mechanical unfolding of human telomere G-quadruplex DNA probed by integrated fluorescence and magnetic tweezers spectroscopy.

Single-molecule techniques facilitate analysis of mechanical transitions within nucleic acids and proteins. Here, we describe an integrated fluorescence and magnetic tweezers instrument that permits detection of nanometer-scale DNA structural rearrangements together with the application of a wide range of stretching forces to individual DNA molecules. We have analyzed the force-dependent equilibrium and rate constants for telomere DNA G-quadruplex (GQ) folding and unfolding, and have determined the location of the transition state barrier along the well-defined DNA-stretching reaction coordinate. Our results reveal the mechanical unfolding pathway of the telomere DNA GQ is characterized by a short distance (<1 nm) to the transition state for the unfolding reaction. This mechanical unfolding response reflects a critical contribution of long-range interactions to the global stability of the GQ fold, and suggests that telomere-associated proteins need only disrupt a few base pairs to destabilize GQ structures. Comparison of the GQ unfolded state with a single-stranded polyT DNA revealed the unfolded GQ exhibits a compacted non-native conformation reminiscent of the protein molten globule. We expect the capacity to interrogate macromolecular structural transitions with high spatial resolution under conditions of low forces will have broad application in analyses of nucleic acid and protein folding.

Single-Molecule Studies of Telomeres and Telomerase.

Telomeres are specialized chromatin structures that protect chromosome ends from dangerous processing events. In most tissues, telomeres shorten with each round of cell division, placing a finite limit on cell growth. In rapidly dividing cells, including the majority of human cancers, cells bypass this growth limit through telomerase-catalyzed maintenance of telomere length. The dynamic properties of telomeres and telomerase render them difficult to study using ensemble biochemical and structural techniques. This review describes single-molecule approaches to studying how individual components of telomeres and telomerase contribute to function. Single-molecule methods provide a window into the complex nature of telomeres and telomerase by permitting researchers to directly visualize and manipulate the individual protein, DNA, and RNA molecules required for telomere function. The work reviewed in this article highlights how single-molecule techniques have been utilized to investigate the function of telomeres and telomerase.

Coordinated DNA dynamics during the human telomerase catalytic cycle.

The human telomerase reverse transcriptase (hTERT) utilizes a template within the integral RNA subunit (hTR) to direct extension of telomeres. Telomerase exhibits repeat addition processivity (RAP) and must therefore translocate the nascent DNA product into a new RNA:DNA hybrid register to prime each round of telomere repeat synthesis. Here, we use single-molecule FRET and nuclease protection assays to monitor telomere DNA structure and dynamics during the telomerase catalytic cycle. DNA translocation during RAP proceeds through a previously uncharacterized kinetic substep during which the 3'-end of the DNA substrate base pairs downstream within the hTR template. The rate constant for DNA primer realignment reveals this step is not rate limiting for RAP, suggesting a second slow conformational change repositions the RNA:DNA hybrid into the telomerase active site and drives the extrusion of the 5'-end of the DNA primer out of the enzyme complex.

Human acellular dermis versus no acellular dermis in tissue expansion breast reconstruction.

BACKGROUND: Human acellular dermis has been adopted for routine use in tissue expander reconstruction. The purported benefits include higher intraoperative fill volume, facilitation of lower pole expansion, and enhanced definition of the lower pole of the breast. Recently, concerns have arisen about an increase in postoperative complications with its use. METHODS: A retrospective review was conducted of patients who had immediate postmastectomy breast reconstruction with a tissue expander from July of 2001 to July of 2011. All tissue expander reconstructions before 2005 were performed submuscularly only and all subsequent to 2005 with the use of AlloDerm (LifeCell, Branchburg, N.J.) acellular dermis. Patient demographics were collected, and complications were recorded. RESULTS: The study cohort included 346 patients and 511 immediate breast reconstructions; 232 patients and 346 breasts were reconstructed with and 114 patients and 165 breasts without acellular dermis. Age, body mass index, diabetes, and tobacco use were equivalent in the two groups. Seroma occurrence in the acellular dermis group was nearly twice (30.0 versus 15.1 percent) that of the no acellular dermis breasts, but the tissue expander loss was only slightly higher (11.6 versus 8.5 percent) and not statistically significant. Body mass index in patients who lost their tissue expander was 31 kg/m, statistically significantly higher than in those who did not. CONCLUSIONS: The presence of acellular dermis did not increase the incidence of tissue expander loss, despite a doubling of frequency of seroma. Prior radiation and use of acellular dermis did culminate in a prohibitively high loss rate of the tissue expander.