Structural basis for DNA strand separation by a hexameric replicative helicase

Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5′ ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted ‘steric exclusion’ model for dsDNA unwinding, the active 3′ ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5′ passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases

Structure of the hDmc1-ssDNA filament reveals the principles of its architecture

In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination

Meloscaphander grandis (Heterobranchia: Cephalaspidea), a deep-water species from the North Pacific: Redescription and taxonomic remarks

Chaban, Elena M., Ekimova, Irina A., Schepetov, Dimitry M., Chernyshev, Alexei V. (2019): Meloscaphander grandis (Heterobranchia: Cephalaspidea), a deep-water species from the North Pacific: Redescription and taxonomic remarks. Zootaxa 4646 (2): 385-400, DOI: https://doi.org/10.11646/zootaxa.4646.2.1

Salt Stress-Induced Structural Changes Are Mitigated in Transgenic Tomato Plants Over-Expressing Superoxide Dismutase

Various abiotic stresses cause the appearance of reactive oxygen species (ROS) in plant cells, which seriously damage the cellular structures. The engineering of transgenic plants with higher production of ROS-scavenging enzyme in plant cells could protect the integrity of such a fine intracellular structure as the cytoskeleton and each cellular compartment. We analyzed the morphological changes in root tip cells caused by the application of iso-osmotic NaCl and Na2SO4 solutions to tomato plants harboring an introduced superoxide dismutase gene. To study the roots of tomato plants cultivar Belyi Naliv (WT) and FeSOD-transgenic line, we examined the distribution of ROS and enzyme-linked immunosorbent detection of α-tubulin. In addition, longitudinal sections of the root apexes were compared. Transmission electronic microscopy of atypical cytoskeleton structures was also performed. The differences in the microtubules cortical network between WT and transgenic plants without salt stress were detected. The differences were found in the cortical network of microtubules between WT and transgenic plants in the absence of salt stress. While an ordered microtubule network was revealed in the root cells of WT tomato, no such degree of ordering was detected in transgenic line cells. The signs of microtubule disorganization in root cells of WT plants were manifested under the NaCl treatment. On the contrary, the cytoskeleton structural organization in the transgenic line cells was more ordered. Similar changes, including the cortical microtubules disorganization, possibly associated with the formation of atypical tubulin polymers as a response to salt stress caused by Na2SO4 treatment, were also observed. Changes in cell size, due to both vacuolization and impaired cell expansion in columella zone and cap initials, were responsible for the root tip tissue modification

Characteristics of Root Cells during In Vitro Rhizogenesis under Action of NaCl in Two Tomato Genotypes Differing in Salt Tolerance

Understanding the mechanisms of plant salt tolerance as a complex trait is an integral part of many studies, the results of which have been used in the breeding process. The aim of this study was to compare the root response of two tomato (Solanum lycopersicum L.) genotypes (breeding line YaLF and cultivar Recordsmen) differing in salt tolerance. Rhizogenesis was induced in tomato shoots in vitro with different concentrations of NaCl in the culture medium. A number of morphobiological and cytological parameters were evaluated at the organ, tissue, and cellular levels for possible use in a comprehensive assessment of genotypes for salt tolerance. The influence of NaCl caused disruption of the cell cycle and redistribution of cells in the phases of the cell cycle. An increase in the degree of vacuolization was shown in cv Recordsmen at 75 and 150 mM NaCl and in the YaLF line at 150 mM NaCl. Under salt action, an increase/decrease in the length of cells such as columella cells (both genotypes) and epidermal cells (in cv Recordsmen at 75 and 150 mM NaCl) was shown. Differences between genotypes were demonstrated by changes in the area of the central cylinder and primary root cortex cells, as well as by changes of the Snucleolus/Snucleus ratio in these cells. Transmission electron microscopy (TEM) showed the modification of the chromatin structure in the root cells of these genotypes. Various cytoskeletal disorders were revealed in interphase cells of the tomato root of cv Recordsmen and the YaLF line by immunofluorescent staining under saline conditions. These morphometric and cytological parameters can be used for a comparative evaluation of genotypes differing in salt tolerance in a comprehensive assessment of varieties