Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication

Treslin, a TopBP1-interacting protein, is necessary for deoxyribonucleic acid (DNA) replication in vertebrates. Association between Treslin and TopBP1 requires cyclin-dependent kinase (Cdk) activity in Xenopus laevis egg extracts. We investigated the mechanism and functional importance of Cdk for this interaction using both X. laevis egg extracts and human cells. We found that Treslin also associated with TopBP1 in a Cdk-regulated manner in human cells and that Treslin was phosphorylated within a conserved Cdk consensus target sequence (on S976 in X. laevis and S1000 in humans). Recombinant human Cdk2–cyclin E also phosphorylated this residue of Treslin in vitro very effectively. Moreover, a mutant of Treslin that cannot undergo phosphorylation on this site showed significantly diminished binding to TopBP1. Finally, human cells harboring this mutant were severely deficient in DNA replication. Collectively, these results indicate that Cdk-mediated phosphorylation of Treslin during S phase is necessary for both its effective association with TopBP1 and its ability to promote DNA replication in human cells

Ataxia-telangiectasia Mutated (ATM)-dependent Activation of ATR Occurs through Phosphorylation of TopBP1 by ATM

ATM (ataxia-telangiectasia mutated) is necessary for activation of Chk1 by ATR (ATM and Rad3-related) in response to double-stranded DNA breaks (DSBs) but not to DNA replication stress. TopBP1 has been identified as a direct activator of ATR. We show that ATM regulates Xenopus TopBP1 by phosphorylating Ser-1131 and thereby strongly enhancing association of TopBP1 with ATR. Xenopus egg extracts containing a mutant of TopBP1 that cannot be phosphorylated on Ser-1131 are defective in the ATR-dependent phosphorylation of Chk1 in response to DSBs but not to DNA replication stress. Thus, TopBP1 is critical for the ATM-dependent activation of ATR following production of DSBs in the genome

Roles of replication fork-interacting and Chk1-activating domains from claspin in a DNA replication checkpoint response

Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. Claspin associates with replication forks upon origin unwinding. We show that Claspin contains a replication fork-interacting domain (RFID, residues 265–605) that associates with Cdc45, DNA polymerase ε, replication protein A, and two replication factor C complexes on chromatin. The RFID contains two basic patches (BP1 and BP2) at amino acids 265–331 and 470–600, respectively. Deletion of either BP1 or BP2 compromises optimal binding of Claspin to chromatin. Absence of BP1 has no effect on the ability of Claspin to mediate activation of Chk1. By contrast, removal of BP2 causes a large reduction in the Chk1-activating potency of Claspin. We also find that Claspin contains a small Chk1-activating domain (residues 776–905) that does not bind stably to chromatin, but it is fully effective at high concentrations for mediating activation of Chk1. These results indicate that stable retention of Claspin on chromatin is not necessary for activation of Chk1. Instead, our findings suggest that only transient interaction of Claspin with replication forks potentiates its Chk1-activating function. Another implication of this work is that stable binding of Claspin to chromatin may play a role in other functions besides the activation of Chk1

Identification of glycosylated marker proteins of epithelial polarity in MDCK cells by homology driven proteomics

BACKGROUND: MDCK cells derived from canine kidney are an important experimental model system for investigating epithelial polarity in mammalian cells. Monoclonal antibodies against apical gp114 and basolateral p58 have served as important tools in these studies. However, the molecular identity of these membrane glycoproteins has not been known. RESULTS: We have identified the sialoglycoprotein gp114 as a dog homologue of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Gp114 was enriched from tissue culture cells by subcellular fractionation and immunoaffinity chromatography. The identification was based on tandem mass spectrometry and homology based proteomics. In addition, the p58 basolateral marker glycoprotein was found to be the β subunit of Na(+)K(+)-ATPase. CONCLUSION: Gp114 has been characterized previously regarding glycosylation dependent trafficking and lipid raft association. The identification as a member of the canine CEACAM family will enable synergy between the fields of epithelial cell biology and other research areas. Our approach exemplifies how membrane proteins can be identified from species with unsequenced genomes by homology based proteomics. This approach is applicable to any model system

Structural and species diversity on North and South slopes in coniferous-deciduous forests of the North-Western Caucasus

The research was conducted in similar soil-orographic conditions of three widespread coniferous-deciduous forest types in the North-Western Caucasus: aspen-hornbeam, beech-fir-hornbeam, and fir-beech. The methods used included geobotanical, population-ontogenetic, and soil-zoological. It was found that the north and south slopes in all the studied forest types, in comparison with the flat areas, are characterised by significantly higher plant species richness. The efficiency of the renewal of arboral cenopopulations was much higher on the slopes, in comparison with the flat areas: the number of tree species (higher by 20–70%); density of tree species (higher by 50–100%); the number of cenopopulations of tree species with a complete ontogenetic spectrum was larger on the slopes, in comparison with the flat areas. A greater number of ecological groups of plants were described on the north and south slopes, in comparison with the flat areas. The proportion of boreal species was higher on the north slopes, in comparison with the south slopes, whereas the majority of meadow-forest edge species were the south slopes. The north and south slopes were characterised by the full functional diversity of soil invertebrates, whereas endemic and sub-endemic macrofauna also preserved. On the north slopes, we revealed a high biomass of large litter and soil saprophages, including species and groups actively participating in transformation of plant litter and soil formation. The south slopes showed high densities of phytophages and predators (among soil macrofauna), which regulate the diversity of other components of forest communities through biotic interactions. All the studied forest types on the north and south slopes exceeded the flat areas in terms of species diversity of plant communities, regeneration of tree species, ecological plant groups, endemic fauna of soil invertebrates, including soil formers. This confirms need to the protection of such territories to preserve and maintain the biological diversity of the coniferous-deciduous forests in the North-Western Caucasus

Education System in Subcarpathian Rus during Interwar Period in the Estimates of the Rusyn Politicians and Public Figures

The article analyzes origins and further development of the education system in Subcarpathian Rus after the incorporation of that region into Czechoslovak republic in 1919 as well as the attitudes of the Rusyn politicians and public figures towards various aspects of school system in Subcarpathian Rus. Since during 1920-ties the school system in Subcarpathian region reflected the policy of “soft ukrainization” of the local Rusyn population pursued by Prague administration in cultural sphere, it aroused growing criticism from Russophile part of Rusyn public and political spectrum while the representatives of Ukrainian movement in Subcarpathian region insisted on more resolute pro-Ukrainian policy in the field of education. Rise of the political and cultural confrontation between the Russophile and Ukrainian intelligentsia of the Carpathian Rusyns became one of the important reasons for the destabilization of the situation in that region in the late 1930s

Charting the protein complexome in yeast by mass spectrometry

It has become evident over the past few years that many complex cellular processes, including control of the cell cycle and ubiquitin-dependent proteolysis, are carried out by sophisticated multisubunit protein machines that are dynamic in abundance, post-translational modification state, and composition. To understand better the nature of the macromolecular assemblages that carry out the cell cycle and ubiquitin-dependent proteolysis, we have used mass spectrometry extensively over the past few years to characterize both the composition of various protein complexes and the modification states of their subunits. In this article we review some of our recent efforts, and describe a promising new approach for using mass spectrometry to dissect protein interaction networks