30 research outputs found
Tough Hydroxyapatite Hydrogels Based on Bone-like Self-Regulatory Sacrificial Bond Formation
Learning
from nature is a promising approach to achieving specific
functions of synthetic materials. The high material functions, in
turn, shed light on the fundamental mechanisms underlying the high
performance of biological tissues. For instance, bone is an attractive
metabolic tissue with fascinating capabilities from the perspective
of both biochemical and biomechanical functionalities. Bone tissue
exhibits exceptional mechanical performance as a skeleton, enabling
to sustain the locomotion of mammals. In this study, we design coupled
reactions for self-regulatory sacrificial bond formation in poly(acrylic
acid) hydrogels by introducing biomineral hydroxyapatite (HAp) found
in bones. We demonstrate that through five coupled reactions, HAp
regulates the Ca2+ bridging to the acidic polymer and toughens
the hydrogels in water by the sacrificial bonds effect. This work
is expected not only to greatly contribute to the design of tough
soft materials but also to give deep insights into the self-regulated
bone-toughening mechanisms
Dynamic Assembly/Disassembly Processes of Photoresponsive DNA Origami Nanostructures Directly Visualized on a Lipid Membrane Surface
Here, we report the direct visualization
of the assembly/disassembly
processes of photoresponsive DNA origami nanostructures which can
be placed on a lipid bilayer surface. The observation relies on controlled
interactions between the bilayer components and cholesterol moieties
introduced to the hexagonal origami structures, one of whose outer
edges carries Azo-ODNs. The bilayer-placed hexagonal dimer was disassembled
into monomer units by UV irradiation, and reversibly assembled again
during visible light irradiation. These dynamic processes were directly
monitored with high-speed atomic force microscopy. The successful
application of our approach should facilitate studies of interactive
and functional behaviors of various DNA nanostructures
MOESM1 of Waveform inversion for 3-D S-velocity structure of D′′ beneath the Northern Pacific: possible evidence for a remnant slab and a passive plume
Additional file 1. Details and validity checks
DNA Origami Based Visualization System for Studying Site-Specific Recombination Events
Site-specific
recombination involves reciprocal exchange between
defined DNA sites. The reaction initiates from the formation of a
recombinase–DNA synaptic complex, in which two recombination
sites arrange in an appropriate configuration. However, there is incomplete
information about how the topological state of the substrate influences
the synapsis and outcome of the reaction. Here, we show that Cre-mediated
recombination can be regulated by controlling the orientation and
topology of the <i>loxP</i> substrate in a DNA frame nanoscaffold.
High-speed atomic force microscopy analyses revealed that the <i>loxP</i>-containing substrate strands in the antiparallel orientation
can be recombined only through formation of synaptic complexes. By
tethering Holliday junction (HJ) intermediates to DNA frames in different
connection patterns and using them as a starting substrate, we found
that the topological state of the HJ intermediates dictates the outcome
of the resolution. Our approach should provide a new platform for
structural–functional studies of various DNA targeting enzymes,
especially which require formation of synaptic complexes
DNA Origami Based Visualization System for Studying Site-Specific Recombination Events
Site-specific
recombination involves reciprocal exchange between
defined DNA sites. The reaction initiates from the formation of a
recombinase–DNA synaptic complex, in which two recombination
sites arrange in an appropriate configuration. However, there is incomplete
information about how the topological state of the substrate influences
the synapsis and outcome of the reaction. Here, we show that Cre-mediated
recombination can be regulated by controlling the orientation and
topology of the <i>loxP</i> substrate in a DNA frame nanoscaffold.
High-speed atomic force microscopy analyses revealed that the <i>loxP</i>-containing substrate strands in the antiparallel orientation
can be recombined only through formation of synaptic complexes. By
tethering Holliday junction (HJ) intermediates to DNA frames in different
connection patterns and using them as a starting substrate, we found
that the topological state of the HJ intermediates dictates the outcome
of the resolution. Our approach should provide a new platform for
structural–functional studies of various DNA targeting enzymes,
especially which require formation of synaptic complexes
Characterization of the Holliday Junction Resolving Enzyme Encoded by the <em>Bacillus subtilis</em> Bacteriophage SPP1
<div><p>Recombination-dependent DNA replication, which is a central component of viral replication restart, is poorly understood in Firmicutes bacteriophages. Phage SPP1 initiates unidirectional theta DNA replication from a discrete replication origin (<em>ori</em>L), and when replication progresses, the fork might stall by the binding of the origin binding protein G<em>38</em>P to the late replication origin (<em>ori</em>R<em>).</em> Replication restart is dependent on viral recombination proteins to synthesize a linear head-to-tail concatemer, which is the substrate for viral DNA packaging. To identify new functions involved in this process, uncharacterized genes from phage SPP1 were analyzed. Immediately after infection, SPP1 transcribes a number of genes involved in recombination and replication from <em>P</em><sub>E2</sub> and <em>P</em><sub>E3</sub> promoters. Resequencing the region corresponding to the last two hypothetical genes transcribed from the <em>P</em><sub>E2</sub> operon (genes <em>44</em> and <em>45</em>) showed that they are in fact a single gene, re-annotated here as gene <em>44</em>, that encodes a single polypeptide, named gene <em>44</em> product (G<em>44</em>P, 27.5 kDa). G<em>44</em>P shares a low but significant degree of identity in its C-terminal region with virus-encoded RusA-like resolvases. The data presented here demonstrate that G<em>44</em>P, which is a dimer in solution, binds with high affinity but without sequence specificity to several double-stranded DNA recombination intermediates. G<em>44</em>P preferentially cleaves Holliday junctions, but also, with lower efficiency, replicated D-loops. It also partially complemented the loss of RecU resolvase activity in <em>B. subtilis</em> cells. These <em>in vitro</em> and <em>in vivo</em> data suggest a role for G<em>44</em>P in replication restart during the transition to concatemeric viral replication.</p> </div
Determination of the cleavage ability of G<i>44</i>P on static and mobile HJs.
<p>(A and C) A fixed HJ (HJ-23M, in A) or a mobile HJ containing a 13-bp homologous core (HJ-jbm6, in C) [γ<sup>32</sup>P]-labeled at the indicated strand was incubated with 10 nM G<i>44</i>P in buffer B containing 10 mM MgCl<sub>2</sub> for 30 min at 37°C. Reaction products were analyzed using 15% denaturing PAGE in the presence (+) or absence (-) of protein. “m” indicates the G+A sequencing ladder obtained for the corresponding labeled oligonucleotide. To serve as additional molecular weight markers, and denoted by C, 41-nt, 21-nt, 23-nt and 18-nt primers were loaded. In lane 13 of panel A, a degraded 17-M oligonucleotide was loaded. (B and D) The cleavage sites detected are indicated by arrows in the core of the two HJ sequences.</p
G<i>44</i>P-mediated cleavage of replicated D-loops.
<p>Different D-loop variants resembling several recombination intermediates were end-labeled at the 5′end of oligonucleotide 19-M (the invading strand, in A) or of oligonucleotide 17-M (the displaced strand, in B) and were incubated with 10 nM G<i>44</i>P in buffer B containing 10 mM MgCl<sub>2</sub> for 30 min at 37°C. Reaction products were analyzed using 20% denaturing PAGE and revealed by autoradiography. Drawings indicate the different substrates analyzed (D-loops A to F, and control HJ and ssDNA). Asterisks indicate the [γ<sup>32</sup>P]-ATP labeling of oligonucleotides at the 5′end. In the HJ substrate, an arrow indicates the major cleavage site. As markers, the G+A sequencing ladder obtained for the corresponding labeled oligonucleotide and the 41-nt and 21-nt primers for the corresponding sequence were loaded.</p
DNA Origami Based Visualization System for Studying Site-Specific Recombination Events
Site-specific
recombination involves reciprocal exchange between
defined DNA sites. The reaction initiates from the formation of a
recombinase–DNA synaptic complex, in which two recombination
sites arrange in an appropriate configuration. However, there is incomplete
information about how the topological state of the substrate influences
the synapsis and outcome of the reaction. Here, we show that Cre-mediated
recombination can be regulated by controlling the orientation and
topology of the <i>loxP</i> substrate in a DNA frame nanoscaffold.
High-speed atomic force microscopy analyses revealed that the <i>loxP</i>-containing substrate strands in the antiparallel orientation
can be recombined only through formation of synaptic complexes. By
tethering Holliday junction (HJ) intermediates to DNA frames in different
connection patterns and using them as a starting substrate, we found
that the topological state of the HJ intermediates dictates the outcome
of the resolution. Our approach should provide a new platform for
structural–functional studies of various DNA targeting enzymes,
especially which require formation of synaptic complexes
G<i>44</i>P binding to different DNA substrates.
<p>EMSAs showing binding of G<i>44</i>P to the indicated [γ<sup>32</sup>P]-labeled DNA substrates: (A) HJ-J3, (B) D-loop DL-D, (C) 80-bp dsDNA, and (D) 80-nt ssDNA. DNA (0.2 nM) was incubated with increasing amounts of G<i>44</i>P as indicated in buffer B containing 1 mM EDTA for 20 min at 37°C. The three types of complexes formed are denoted by I, II, and III.</p