Human RIF1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation

We thank David Stead at the Aberdeen Proteomics Service for help in mass spectrometry interpretation, and Raif Yücel and his team at the University of Aberdeen Iain Fraser Cytometry Centre for assistance with flow cytometry. We thank Robert Alver and Julian Blow at University of Dundee for advice on the use of tautomycetin. Peter Cherepanov of the Francis Crick Institute gifted XL413. Daniel Durocher of Lunenfeld-Tanenbaum Research Institute gifted DNA constructs. Work by ADD and SH was supported by Cancer Research UK Grant A13356, Cancer Research UK Programme Award A19059, and BBSRC grant (BB/K006304/1). AIL was supported by Wellcome Trust Awards (108058/Z/15/Z & 105024/Z/14/Z). This work was also supported by JSPS KAKENHI Grant # 16H04739, 25116004 to CO and 16J04327 to YO.Peer reviewedPublisher PD

γ-Tubulin 2 Nucleates Microtubules and Is Downregulated in Mouse Early Embryogenesis

γ-Tubulin is the key protein for microtubule nucleation. Duplication of the γ-tubulin gene occurred several times during evolution, and in mammals γ-tubulin genes encode proteins which share ∼97% sequence identity. Previous analysis of Tubg1 and Tubg2 knock-out mice has suggested that γ-tubulins are not functionally equivalent. Tubg1 knock-out mice died at the blastocyst stage, whereas Tubg2 knock-out mice developed normally and were fertile. It was proposed that γ-tubulin 1 represents ubiquitous γ-tubulin, while γ-tubulin 2 may have some specific functions and cannot substitute for γ-tubulin 1 deficiency in blastocysts. The molecular basis of the suggested functional difference between γ-tubulins remains unknown. Here we show that exogenous γ-tubulin 2 is targeted to centrosomes and interacts with γ-tubulin complex proteins 2 and 4. Depletion of γ-tubulin 1 by RNAi in U2OS cells causes impaired microtubule nucleation and metaphase arrest. Wild-type phenotype in γ-tubulin 1-depleted cells is restored by expression of exogenous mouse or human γ-tubulin 2. Further, we show at both mRNA and protein levels using RT-qPCR and 2D-PAGE, respectively, that in contrast to Tubg1, the Tubg2 expression is dramatically reduced in mouse blastocysts. This indicates that γ-tubulin 2 cannot rescue γ-tubulin 1 deficiency in knock-out blastocysts, owing to its very low amount. The combined data suggest that γ-tubulin 2 is able to nucleate microtubules and substitute for γ-tubulin 1. We propose that mammalian γ-tubulins are functionally redundant with respect to the nucleation activity

Additional Precipitation Reactions of Lectins with Human Serum Glycoproteins

Peer Reviewe

Protein-Engineered Proteinase of Myeloblastosis Associated Virus, An Enzyme of High Activity and HIV-1 Proteinase-Like Specificity

All proteinases of avian and mammalian retroviruses belong to the family of aspartic proteinases, are of similar size and of homologous primary structure; they all act catalytically in the form of highly symmetric molecular dimers.1 Detailed studies of retroviral proteinases were carried out on two almost identical proteinases of MAV2,3 and RS V4 (representing the group of avian retroviruses) and on the HIV proteinase.5,6 The knowledge of the 3D structure,2,4,5 catalytic activity and substrate specifity3,6 of the MAV and the HIV proteinase has changed the notion of their general similarity since several features that distinguish each proteinase from the other were revealed. The HIV-1 proteinase has a considerably higher activity3,6 which reflects the different conditions of the expression and action of this enzyme in vivo: 7 The “coding strategy” of MAV allows the expression of the proteinase from the first (gag) open reading frame and provides for the high (i.e. stoichiometrical) level of the relatively “weak” enzyme whereas the smaller amount of the more active HIV enzyme is a result of infrequent translational frameshift events that occur in the overlapping region of the gag and pol reading frames.8 The substrate specificities of retroviral proteinases seem complex and the requirement for a side chain in an individual subsite of a substrate is an outcome of the combination of residues occupying other closely located subsites.3 The two proteinases (MAV and HIV) show rather promiscuous substrate specificity, nevertheless several differences can be traced. We made an attempt to use protein engineering of the MAV proteinase to tackle directly problems of structural basis of these differences and, vice versa, to make more precise conclusions on the functional importance of the individual elements of its three dimensional structure. This article describes mutation of the MAV proteinase which resulted not only in an alteration of its substrate specificity but also in an increase of its enzymic activity — a rare case in protein engineering