Three-dimensional flagella structures from animals' closest unicellular relatives, the Choanoflagellates.

In most eukaryotic organisms, cilia and flagella perform a variety of life-sustaining roles related to environmental sensing and motility. Cryo-electron microscopy has provided considerable insight into the morphology and function of flagellar structures, but studies have been limited to less than a dozen of the millions of known eukaryotic species. Ultrastructural information is particularly lacking for unicellular organisms in the Opisthokonta clade, leaving a sizeable gap in our understanding of flagella evolution between unicellular species and multicellular metazoans (animals). Choanoflagellates are important aquatic heterotrophs, uniquely positioned within the opisthokonts as the metazoans' closest living unicellular relatives. We performed cryo-focused ion beam milling and cryo-electron tomography on flagella from the choanoflagellate species Salpingoeca rosetta. We show that the axonemal dyneins, radial spokes, and central pair complex in S. rosetta more closely resemble metazoan structures than those of unicellular organisms from other suprakingdoms. In addition, we describe unique features of S. rosetta flagella, including microtubule holes, microtubule inner proteins, and the flagellar vane: a fine, net-like extension that has been notoriously difficult to visualize using other methods. Furthermore, we report barb-like structures of unknown function on the extracellular surface of the flagellar membrane. Together, our findings provide new insights into choanoflagellate biology and flagella evolution between unicellular and multicellular opisthokonts.

Photochemical modifications for DNA/RNA oligonucleotides.

Light-triggered chemical reactions can provide excellent tools to investigate the fundamental mechanisms important in biology. Light is easily applicable and orthogonal to most cellular events, and its dose and locality can be controlled in tissues and cells. Light-induced conversion of photochemical groups installed on small molecules, proteins, and oligonucleotides can alter their functional states and thus the ensuing biological events. Recently, photochemical control of DNA/RNA structure and function has garnered attention thanks to the rapidly expanding photochemistry used in diverse biological applications. Photoconvertible groups can be incorporated in the backbone, ribose, and nucleobase of an oligonucleotide to undergo various irreversible and reversible light-induced reactions such as cleavage, crosslinking, isomerization, and intramolecular cyclization reactions. In this review, we gather a list of photoconvertible groups used in oligonucleotides and summarize their reaction characteristics, impacts on DNA/RNA thermal stability and structure, as well as their biological applications.

Impact of DNA sequences on DNA 'opening' by the Rad4/XPC nucleotide excision repair complex.

Rad4/XPC recognizes diverse DNA lesions to initiate nucleotide excision repair (NER). However, NER propensities among lesions vary widely and repair-resistant lesions are persistent and thus highly mutagenic. Rad4 recognizes repair-proficient lesions by unwinding ('opening') the damaged DNA site. Such 'opening' is also observed on a normal DNA sequence containing consecutive C/G's (CCC/GGG) when tethered to Rad4 to prevent protein diffusion. However, it was unknown if such tethering-facilitated DNA 'opening' could occur on any DNA or if certain structures/sequences would resist being 'opened'. Here, we report that DNA containing alternating C/G's (CGC/GCG) failed to be opened even when tethered; instead, Rad4 bound in a 180°-reversed manner, capping the DNA end. Fluorescence lifetime studies of DNA conformations in solution showed that CCC/GGG exhibits local pre-melting that is absent in CGC/GCG. In MD simulations, CGC/GCG failed to engage Rad4 to promote 'opening' contrary to CCC/GGG. Altogether, our study illustrates how local sequences can impact DNA recognition by Rad4/XPC and how certain DNA sites resist being 'opened' even with Rad4 held at that site indefinitely. The contrast between CCC/GGG and CGC/GCG sequences in Rad4-DNA recognition may help decipher a lesion's mutagenicity in various genomic sequence contexts to explain lesion-determined mutational hot and cold spots.

Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein.

Biomolecular structural changes upon binding/unbinding are key to their functions. However, characterization of such dynamical processes is difficult as it requires ways to rapidly and specifically trigger the assembly/disassembly as well as ways to monitor the resulting changes over time. Recently, various chemical strategies have been developed to use light to trigger changes in oligonucleotide structures, and thereby their activities. Here we report that photocleavable DNA can be used to modulate the DNA binding of the Rad4/XPC DNA repair complex using light. Rad4/XPC specifically recognizes diverse helix-destabilizing/distorting lesions including bulky organic adduct lesions and functions as a key initiator for the eukaryotic nucleotide excision repair (NER) pathway. We show that the 6-nitropiperonyloxymethyl (NPOM)-modified DNA is recognized by the Rad4 protein as a specific substrate and that the specific binding can be abolished by light-induced cleavage of the NPOM group from DNA in a dose-dependent manner. Fluorescence lifetime-based analyses of the DNA conformations suggest that free NPOM-DNA retains B-DNA-like conformations despite its bulky NPOM adduct, but Rad4-binding causes it to be heterogeneously distorted. Subsequent extensive conformational searches and molecular dynamics simulations demonstrate that NPOM in DNA can be housed in the major groove of the DNA, with stacking interactions among the nucleotide pairs remaining largely unperturbed and thus retaining overall B-DNA conformation. Our work suggests that photoactivable DNA may be used as a DNA lesion surrogate to study DNA repair mechanisms such as nucleotide excision repair.

Tethering-facilitated DNA 'opening' and complementary roles of ß-hairpin motifs in the Rad4/XPC DNA damage sensor protein.

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively 'opening' these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a 'kinetic gating' mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how 'opening' is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed 'open' undamaged DNA in solution and that such 'opening' can largely occur without one or the other of the ß-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound 'open' DNA adopts multiple conformations in solution notwithstanding the DNA's original structure or the ß-hairpins. Molecular dynamics simulations reveal compensatory roles of the ß-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA 'opening' of undamaged sites and the dynamic nature of 'open' DNA may shed light on how the protein functions within and beyond nucleotide excision repair in cells.

Ultrasonic-assisted synthesis of Ce doped cubic-hexagonal ZnTiO3 with highly efficient sonocatalytic activity.

Ce doped ZnTiO3 as a novel catalyst with highly efficient and stable sonocatalytic activity was synthesized via an ultrasound-assisted sol-gel method using non-ionic surfactant Pluronic F127 as structure directing agent. Synthesized samples were characterized by using various techniques, such as XRD, TEM, SEM, EDX, ​XRF, BET, DRS, and PL, and their sonocatalytic activity studied toward degradation of p-Nitrophenol as a model organic compound. The synthesized mesoporous Ce/ZnTiO3 had mixed cubic-hexagonal phase with large surface area (118.2 m(2) g(-1)) and narrow pore size distribution (4.9 nm). The effects of cerium concentration, calcination temperature, and calcination time on the structure and the sonocatalytic activity of Ce/ZnTiO3 were studied in detail. XRD results were suggested that the relation between the phase structure and the catalytic activity is considerable. Significant decrease in band-gap and PL intensity was observed with increasing the cerium concentration in the ZnTiO3. It became clear that the Ce/ZnTiO3 (0.81 mol%) shows high sonocatalytic activity compared with pure ZnTiO3 and other Ce/ZnTiO3 samples as well as commercial TiO2-P25. The possible mechanism for the enhanced sonocatalytic activity of Ce/ZnTiO3 was discussed in details. The electrical energy consumption was also considered during sonocatalytic experiments.