TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity

Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase–mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1’s role in HR, with potential clinical implications for cancer treatment.     

Somatic embryogenesis and bud formation from immature embryos of buckwheat (Fagopyrum esculentum Moench.)

Immature embryos of Fagopyrum esculentum cv. Pennquad were isolated from field-grown plants and cultured on media containing a high benzylaminopurine to indole-3-acetic acid ratio. Part of the embryos were grown in the presence of 2,4-dichlorophenoxyacetic acid and kinetin for the first 5 days, and then transferred to benzylaminopurine + indole-3-acetic acid medium. From callus tissues developed on hypocotyls and cotyledons, 3 types of tissue were selected in later subcultures: (a) callus tissue strains that produced buds, (b) embryogenic tissue, and (c) unorganized callus tissue, lacking any organogenic capacity. Pretreatment with 2,4-dichlorophenoxyacetic acid increased the number of explants which gave rise to bud forming and embryogenic tissue, but was not essential for morphogenesis. Somatic embryogenesis was confirmed by histological observation. Plantlets could be easily obtained by inducing adventitious roots on shoots, but spontaneous root development in somatic embryos was infrequent.Abbreviations BAP benzylaminopurine - IAA indole-3-acetic acid - 2,4-D dichlorophenoxyacetic acid - IBA indole-3-butyric acid     

Telomerase RNA in Hymenoptera (Insecta) switched to plant/ciliate-like biogenesis

In contrast to the catalytic subunit of telomerase, its RNA subunit (TR) is highly divergent in size, sequence and biogenesis pathways across eukaryotes. Current views on TR evolution assume a common origin of TRs transcribed with RNA polymerase II in Opisthokonta (the supergroup including Animalia and Fungi) and Trypanosomida on one hand, and TRs transcribed with RNA polymerase III under the control of type 3 promoter, found in TSAR and Archaeplastida supergroups (including e.g. ciliates and Viridiplantae taxa, respectively). Here, we focus on unknown TRs in one of the largest Animalia order - Hymenoptera (Arthropoda) with more than 300 available representative genomes. Using a combination of bioinformatic and experimental approaches, we identify their TRs. In contrast to the presumed type of TRs (H/ACA box snoRNAs transcribed with RNA Polymerase II) corresponding to their phylogenetic position, we find here short TRs of the snRNA type, likely transcribed with RNA polymerase III under the control of the type 3 promoter. The newly described insect TRs thus question the hitherto assumed monophyletic origin of TRs across Animalia and point to an evolutionary switch in TR type and biogenesis that was associated with the divergence of Arthropods.     

The memory enhancing effect of the APP-derived tripeptide Ac-rER is mediated through CRMP2

Photoactivated adenylyl cyclase α (PACα) was originally isolated from the flagellate Euglena gracilis. Following stimulation by blue light it causes a rapid increase in cAMP levels. In the present study, we expressed PACα in cholinergic neurons of Caenorhabditis elegans. Photoactivation led to a rise in swimming frequency, speed of locomotion, and a decrease in the number of backward locomotion episodes. The extent of the light-induced behavioral effects was dependent on the amount of PACα that was expressed. Furthermore, electrophysiological recordings from body wall muscle cells revealed an increase in miniature post-synaptic currents during light stimulation. We conclude that the observed effects were caused by cAMP synthesis because of photoactivation of pre-synaptic PACα which subsequently triggered acetylcholine release at the neuromuscular junction. Our results demonstrate that PACα can be used as an optogenetic tool in C. elegans for straightforward in vivo manipulation of intracellular cAMP levels by light, with good temporal control and high cell specificity. Thus, using PACα allows manipulation of neurotransmitter release and behavior by directly affecting intracellular signaling.     

Benzyladenine induction of buds and somatic embryogenesis in Picea omorika (Pančić) Purk.

Somatic embryogenesis and adventitious bud formation, initiated from shoot explants of Picea omorika is described. Benzyladenine (BA) as the only growth regulator, added to modified Von Arnold and Eriksson medium, induced formation of both adventitious buds and embryogenic tissue. Optimal BA concentration for bud induction was 4.5 M and further bud development and plantlet formation was achieved on growth regulator-free medium. The embryogenic tissue formation was induced when the explants were first grown on the medium with high BA content (22.5 M) and then transferred to medium without growth regulators. Subsequent proliferation of embryogenic tissue was accomplished by subculturing on medium containing 9 M 2,4-dichlorophenoxyacetic acid and 4.5 M BA, and further embryo development was achieved on medium with 12 M abscisic acid. Embryos cultured on growth regulator-free medium formed roots and rooted plantlets were successfully established in soil in the greenhouse.     

Insights into the evolution of mammalian telomerase: Platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes

ABSTRACT: BACKGROUND: The TERT gene encodes the catalytic subunit of the telomerase complex and is responsible for maintaining telomere length. Vertebrate telomerase has been studied in placental mammals, fish, and the chicken, but less attention has been paid to other vertebrates. The platypus occupies an important evolutionary position, providing unique insight into the evolution of mammalian genes. We report the cloning of a platypus TERT (pTERT) ortholog, and provide a comparison with genes of other vertebrates. RESULTS: The pTERT encodes a protein with the high homology to marsupial TERT and avian TERT. Like the TERT of sauropsids and marsupials, as well as that of sharks and echinoderms, pTERT contains extended variable linkers in the N-terminal region suggesting that they were present already in basal vertebrates and lost independently in placental mammals and ray-finned fish. Several alternatively spliced pTERT variants structurally similar to avian TERT variants were identified. Telomerase activity is expressed in all platypus tissues similarly to cold-blooded animals and murine rodents. pTERT was localized on pseudoautosomal regions of sex chromosomes X3/Y2, expanding the homology between human chromosome 5 and platypus sex chromosomes. The synteny analysis suggests that TERT co-localized with sex-linked genes in the last common mammalian ancestor. Interestingly, female platypuses express higher levels of telomerase in heart and liver tissues than do males. CONCLUSIONS: pTERT shares many features with TERT of the reptilian outgroup, suggesting that pTERT represents the ancestral mammalian TERT. Features specific to TERT of eutherian mammals have, therefore, evolved more recently after the divergence of monotremes.     

Synthesis of proteins and RNA of the 60S ribosomal subunit in HeLa cells recovering from valine deprivation

The synthesis of ribosomes in HeLa cells was studied during recovery from a 20-hour deprivation for valine. The rates of incorporation of labeled precursors into ribosomal pre-RNA, processed rRNA, total cellular proteins, and proteins of the 60S ribosomal subunit returned to normal or nearly normal levels immediately after restoration of valine to the medium. Specific proteins of the 60S ribosomal subunit, whose apparent net synthesis is reduced more than that of the other proteins of the 60S ribosomal subunit during valine deprivation, were no longer undersynthesized after valine was restored. This rapid recovery suggests that the apparent decrease in the net rate of synthesis of these ribosomal proteins during valine deprivation is effected at the translational or post-translational level. No evidence of significant synchrony in any particular stage of the cell cycle was observed after a 20-hr valine deprivation. Key words: 60S ribosomal subunit; HeLa, cells; valine deprivation.     

Festucion carpaticae alliancia nova—a new alliance of tall grasslands in the high Carpathian Mountains

For tall grasslands of Carpathian avalanche troughs with dominantFestuca carpatica (orPoa nemoralis subsp.montana) a new alliance ofFestucion carpaticae has been established. AssociationFestucetum carpaticae Domin 1925 has been introduced into the alliance, with some new localities supported by phytocenological relevés. A new subass.senecietosum nemorensis was described from a forest zone. The plant community with dominantFestuca carpatica of the Roumanian Carpathian Mts. has been separated into a new associationSeslerio rigidae-Festucetum carpaticae and further, a new association of the same areaSaxifrago heucherifolii-Poetum montanae is described. Synmorfological, synecological, syntaxonomical and synchorological characteristics of the associations are mentioned.