In Escherichia coli, the primosome plays an essential role in replication restart after dissociation of replisomes at the damaged replication fork. As well as PriA and PriB, DnaT, an ssDNA-binding protein, is a key member of the primosome. In this study, limited proteolysis indicated that E. coli DnaT was composed of two domains, consistent with the results of recent studies using Klebsiella pneumonia DnaT. We also found that a specific 24-residue region (Phe42-Asp66) in the N-terminal domain (1-88) was crucial for DnaT trimerization. Moreover, we determined the structure of the DnaT C-terminal domain (89-179) by NMR spectroscopy. This domain included three a-helices and a long flexible C-terminal tail, similar to the C-terminal subdomain of the AAA+ ATPase family. The neighboring histidines, His136 and His137, at a position corresponding to the AAA+ sensor II motif, were suggested to form an ssDNA-binding site. Furthermore, we found that the acidic linker between the two domains had an activity for dissociating ssDNA from the PriBÁssDNA complexes in a manner supported by the conserved acidic residues Asp70 and Glu76. Thus, these findings provide a novel structural basis for understanding the mechanism of DnaT in exposure of ssDNA and reloading of the replicative helicase at the stalled replication fork.
DatabaseThe coordinates used for the ensemble of NMR structures have been deposited in the Protein Data Bank under accession code 2ru8. The NMR data have been deposited in the BioMagResBank (www.bmrb.wisc.edu) under accession number 11549.
Structured digital abstract• DnaT and DnaT bind by nuclear magnetic resonance (View interaction)• DnaT and PriB bind by isothermal titration calorimetry (View interaction)• DnaT and DnaT bind by molecular sieving (View interaction)Abbreviations EMSA, electrophoresis mobility shift assay; HSQC, heteronuclear single quantum coherence; ITC, isothermal titration calorimetry; PDB, Protein Data Bank.
Renal anemia is predominantly caused by a relative deficiency in erythropoietin (EPO). Conventional treatment for renal anemia includes the use of recombinant human EPO (rhEPO) or a long-acting erythropoiesis-activating agent named darbepoetin alfa, which is a modified rhEPO with a carbohydrate chain structure that differs from native hEPO. We have developed a biosimilar to darbepoetin alfa designated JR-131. Here, we comprehensively compare the physicochemical and biological characteristics of JR-131 to darbepoetin alfa. JR-131 demonstrated similar protein structure to the originator, darbepoetin alfa, by peptide mapping and circular dichroism spectroscopy. Additionally, mass spectroscopic analyses and capillary zone electrophoresis revealed similar glycosylation patterns between the two products. Human bone marrow-derived erythroblasts differentiated and proliferated to form colonies with JR-131 to a similar degree as darbepoetin alfa. Finally, JR-131 stimulated erythropoiesis and improved anemia in rats similarly to darbepoetin alfa. Our data show the similarity in physicochemical and biological properties of JR-131 to those of darbepoetin alfa, and JR-131 therefore represents a biosimilar for use in the treatment of renal anemia.
In this paper, we propose an online probabilistic activation/deactivation control method for base stations (BSs) in heterogeneous networks based on the temporal system throughput and activation states of neighbor BSs (cells). The conventional method iteratively updates the activation/deactivation states in a probabilistic manner at each BS based on the change in the observed system throughput and activation/deactivation states of that BS between past multiple consecutive discrete times. Since BS activation control increases the system throughput by improving the tradeoff between the reduction in inter-cell interference and the traffic off-loading effect, the activation of a BS whose neighbor BSs are deactivated is likely to result in improved system performance and vice versa. The proposed method newly introduces a metric, which represents the effective ratio of the activated neighbor BSs considering their transmission power and distance to the BS of interest, to the update control of the activation probability. This improves both the convergence rate of the iterative algorithm and throughput performance after convergence. Computer simulation results, in which the mobility of the user terminals is taken into account, show the effectiveness of the proposed method.
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