40 research outputs found
The Telomere Capping Complex CST Has an Unusual Stoichiometry, Makes Multipartite Interaction with G-Tails, and Unfolds Higher-Order G-Tail Structures
The telomere-ending binding protein complex CST (Cdc13-Stn1-Ten1) mediates critical functions in both telomere protection and replication. We devised a co-expression and affinity purification strategy for isolating large quantities of the complete Candida glabrata CST complex. The complex was found to exhibit a 2ā¶4ā¶2 or 2ā¶6ā¶2 stoichiometry as judged by the ratio of the subunits and the native size of the complex. Stn1, but not Ten1 alone, can directly and stably interact with Cdc13. In gel mobility shift assays, both Cdc13 and CST manifested high-affinity and sequence-specific binding to the cognate telomeric repeats. Single molecule FRET-based analysis indicates that Cdc13 and CST can bind and unfold higher order G-tail structures. The protein and the complex can also interact with non-telomeric DNA in the absence of high-affinity target sites. Comparison of the DNAāprotein complexes formed by Cdc13 and CST suggests that the latter can occupy a longer DNA target site and that Stn1 and Ten1 may contact DNA directly in the full CSTāDNA assembly. Both Stn1 and Ten1 can be cross-linked to photo-reactive telomeric DNA. Mutating residues on the putative DNAābinding surface of Candida albicans Stn1 OB fold domain caused a reduction in its crosslinking efficiency in vitro and engendered long and heterogeneous telomeres in vivo, indicating that the DNAābinding activity of Stn1 is required for telomere protection. Our data provide insights on the assembly and mechanisms of CST, and our robust reconstitution system will facilitate future biochemical analysis of this important complex
Junction resolving enzymes use multivalency to keep the Holliday junction dynamic
Holliday junction (HJ) resolution by resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single-molecule fluorescence resonance energy transfer analysis of resolving enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.11Nsciescopu
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Developmental mechanism of the periodic membrane skeleton in axons
Actin, spectrin, and associated molecules form a periodic sub-membrane lattice structure in axons. How this membrane skeleton is developed and why it preferentially forms in axons are unknown. Here, we studied the developmental mechanism of this lattice structure. We found that this structure emerged early during axon development and propagated from proximal regions to distal ends of axons. Components of the axon initial segment were recruited to the lattice late during development. Formation of the lattice was regulated by the local concentration of Ī²II spectrin, which is higher in axons than in dendrites. Increasing the dendritic concentration of Ī²II spectrin by overexpression or by knocking out ankyrin B induced the formation of the periodic structure in dendrites, demonstrating that the spectrin concentration is a key determinant in the preferential development of this structure in axons and that ankyrin B is critical for the polarized distribution of Ī²II spectrin in neurites. DOI: http://dx.doi.org/10.7554/eLife.04581.00
Prevalent presence of periodic actin-spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species
Actin, spectrin, and associated molecules form a periodic, submembrane cytoskeleton in the axons of neurons. For a better understanding of this membrane-associated periodic skeleton (MPS), it is important to address how prevalent this structure is in different neuronal types, different subcellular compartments, and across different animal species. Here, we investigated the organization of spectrin in a variety of neuronal- and glial-cell types. We observed the presence of MPS in all of the tested neuronal types cultured from mouse central and peripheral nervous systems, including excitatory and inhibitory neurons from several brain regions, as well as sensory and motor neurons. Quantitative analyses show that MPS is preferentially formed in axons in all neuronal types tested here: Spectrin shows a long-range, periodic distribution throughout all axons but appears periodic only in a small fraction of dendrites, typically in the form of isolated patches in subregions of these dendrites. As in dendrites, we also observed patches of periodic spectrin structures in a small fraction of glial-cell processes in four types of glial cells cultured from rodent tissues. Interestingly, despite its strong presence in the axonal shaft, MPS is disrupted in most presynaptic boutons but is present in an appreciable fraction of dendritic spine necks, including some projecting from dendrites where such a periodic structure is not observed in the shaft. Finally, we found that spectrin is capable of adopting a similar periodic organization in neurons of a variety of animal species, including Caenorhabditis elegans, Drosophila, Gallus gallus, Mus musculus, and Homo sapiens
Fluorescence-force spectroscopy at the single molecule level
During the past decade, various powerful single-molecule techniques have
evolved and helped to address important questions in life sciences. As the single
molecule techniques become mature, there is increasingly pressing need to maximize the
information content of the analysis in order to be able to study more complex systems
that better approximate in-vivo conditions. Here, we develop a fluorescence-force
spectroscopy method to combine single-molecule fluorescence spectroscopy with optical
tweezers. Optical tweezers are used to manipulate and observe mechanical properties on
the nanometer scale and piconewton force range. However, once the force range is in the
low piconewton range or less, the spatial resolution of optical tweezers decreases
significantly. In combination with fluorescence spectroscopy, like single molecule
Fƶrster (or fluorescence) resonance energy transfer (FRET) whose detectable distance
range is approximately 3-10 nm, we are able to observe nanometer fluctuations and
internal conformational changes in a low-force regime. The possibility to place
fluorescent labels at nearly any desired position and a sophisticated design of the
experiment increases the amount of information that can be extracted in contrast to pure
mechanical or fluorescence experiments. We demonstrate the applications of this method
to various biological systems including: 1) to measure the effect of very low forces on the
nanometer scale conformational transitions of the DNA four-way (Holliday) junction; 2)
to dissect protein diffusion and dissociation mechanisms on single stranded DNA, 3) to
calibrate FRET-based in-vivo force sensors and 4) to study mechanical unfolding of
single proteins. The results could not have been obtained with fluorescence or force
measurement alone, and clearly demonstrates the power and generality of our approach.
Finally, we show that self-quenching of two identical fluorophores can be used to detect
small conformational dynamics corresponding to sub-nanometer distance changes of
single molecules in a FRET-insensitive short range (< 3 nm), extending the detectable
distance range of our fluorescence-force spectroscopy method
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Periodic DNA patrolling underlies diverse functions of Pif1 on R-loops and G-rich DNA
Pif1 family helicases are conserved from bacteria to humans. Here, we report a novel
DNA patrolling activity which may underlie Pif1ās diverse functions: a Pif1 monomer preferentially
anchors itself to a 3ā²-tailed DNA junction and periodically reel in the 3ā² tail with a step size of one
nucleotide, extruding a loop. This periodic patrolling activity is used to unfold an intramolecular
G-quadruplex (G4) structure on every encounter, and is sufficient to unwind RNA-DNA heteroduplex
but not duplex DNA. Instead of leaving after G4 unwinding, allowing it to refold, or going beyond
to unwind duplex DNA, Pif1 repeatedly unwinds G4 DNA, keeping it unfolded. Pif1-induced
unfolding of G4 occurs in three discrete steps, one strand at a time, and is powerful enough to
overcome G4-stabilizing drugs. The periodic patrolling activity may keep Pif1 at its site of in vivo
action in displacing telomerase, resolving R-loops, and keeping G4 unfolded during replication,
recombination and repair