Inner centromere formation requires hMis14, a trident kinetochore protein that specifically recruits HP1 to human chromosomes

hMis14 and HP1 depend on each other to localize to the kinetochore and inner centromere, respectively

Protein phosphatase 1 acts as a RIF1 effector to suppress DSB resection prior to Shieldin action

Funding Information: We thank K. Murakami, H. Kimura, H. Kurumizaka, and J.M. Stark for materials. This work was supported by JSPS KAKENHI grant nos. JP19H03156 , JP18H04713 , JP18H05532 , and JP25116004 (to C.O.), JP17H06426 (to K.N.), JP18H04900 and JP19H04267 (to H.S.), the Mitsubishi Foundation (to H.S.), and by Cancer Research UK awards C1445/A19059 and DRCPGM/100013 (to S.H. and A.D.D. lab). Publisher Copyright: © 2021 The Author(s)Peer reviewedPublisher PD

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

BRCA1 Directs the Repair Pathway to Homologous Recombination by Promoting 53BP1 Dephosphorylation

Summary: BRCA1 promotes homologous recombination (HR) by activating DNA-end resection. By contrast, 53BP1 forms a barrier that inhibits DNA-end resection. Here, we show that BRCA1 promotes DNA-end resection by relieving the 53BP1-dependent barrier. We show that 53BP1 is phosphorylated by ATM in S/G2 phase, promoting RIF1 recruitment, which inhibits resection. 53BP1 is promptly dephosphorylated and RIF1 released, despite remaining unrepaired DNA double-strand breaks (DSBs). When resection is impaired by CtIP/MRE11 endonuclease inhibition, 53BP1 phosphorylation and RIF1 are sustained due to ongoing ATM signaling. BRCA1 depletion also sustains 53BP1 phosphorylation and RIF1 recruitment. We identify the phosphatase PP4C as having a major role in 53BP1 dephosphorylation and RIF1 release. BRCA1 or PP4C depletion impairs 53BP1 repositioning, EXO1 recruitment, and HR progression. 53BP1 or RIF1 depletion restores resection, RAD51 loading, and HR in PP4C-depleted cells. Our findings suggest that BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward HR. : Following induction of DNA double-strand break, a pro-end-joining environment is created in G2 by transient 53BP1 phosphorylation and RIF1 recruitment. Here, Isono et al. show that, if timely repair does not ensue, BRCA1 promotes 53BP1 dephosphorylation and RIF1 release, favoring repair by homologous recombination. Keywords: ATM, DNA-end resection, BRCA1, 53BP1, RIF1, PP4C, NHEJ, H

Active establishment of centromeric CENP-A chromatin by RSF complex

Centromeres are chromosomal structures required for equal DNA segregation to daughter cells, comprising specialized nucleosomes containing centromere protein A (CENP-A) histone, which provide the basis for centromeric chromatin assembly. Discovery of centromere protein components is progressing, but knowledge related to their establishment and maintenance remains limited. Previously, using anti-CENP-A native chromatin immunoprecipitation, we isolated the interphase–centromere complex (ICEN). Among ICEN components, subunits of the remodeling and spacing factor (RSF) complex, Rsf-1 and SNF2h proteins, were found. This paper describes the relationship of the RSF complex to centromere structure and function, demonstrating its requirement for maintenance of CENP-A at the centromeric core chromatin in HeLa cells. The RSF complex interacted with CENP-A chromatin in mid-G1. Rsf-1 depletion induced loss of centromeric CENP-A, and purified RSF complex reconstituted and spaced CENP-A nucleosomes in vitro. From these data, we propose the RSF complex as a new factor actively supporting the assembly of CENP-A chromatin

A novel method for purification of the endogenously expressed fission yeast Set2 complex

Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield. Here, we describe a new method for optimized purification of the endogenously expressed fission yeast Set2 complex, histone H3K36 methyltransferase. Using the standard centrifugation procedure for purification, approximately half of the Set2 protein separated into the insoluble chromatin pellet fraction, making it impossible to recover the large amounts of soluble Set2. To overcome this poor recovery, we developed a novel protein purification technique termed the filtration/immunoaffinity purification/mass spectrometry (FIM) method, which eliminates the need for centrifugation. Using the FIM method, in which whole cell lysates were filtered consecutively through eight different pore sizes (53-0.8 mu m), a high yield of soluble FLAG-tagged Set2 was obtained from fission yeast. The technique was suitable for affinity purification and produced a low background. A mass spectrometry analysis of anti-FLAG immunoprecipitated proteins revealed that Rpb1, Rpb2 and Rpb3, which have all been reported previously as components of the budding yeast Set2 complex, were isolated from fission yeast using the FIM method. In addition, other subunits of RNA polymerase II and its phosphatase were also identified. In conclusion, the FIM method is valid for the efficient purification of protein complexes that separate into the insoluble chromatin pellet fraction during centrifugation. (C) 2014 Elsevier Inc. All rights reserved

Characterization of the interaction of influenza virus NS1 with Akt

Avian influenza viruses belong to the genus influenza A virus of the family Orthomyxoviridae. The influenza virus consists of eight segmented minus stranded RNA that encode 11 known proteins. Among the 11 viral proteins, NS1 (non-structural protein 1, encoded on segment 8) has been implicated in the regulation of several important intra-cellular functions. In this report, we investigated the functional interaction of NS1 with serine threonine kinase Akt, a core intra-cellular survival regulator. In co-immunoprecipitation assays and GST pull-down assays, NS1 directly interacted with Akt. The interaction was mediated primarily through the Akt-PH (Pleckstrin Homology) domain and the RNA-binding domain of NS1. NS1 preferentially interacted with phosphorylated Akt, but not with non-phosphorylated Akt. Functionally, the NS1-Akt interaction enhanced Akt kinase activity both in the intra-cellular context and in in vitro Akt kinase assays. Confocal microscopic analysis revealed that phosphorylated Akt interacted with NS1 during the interphase of the cell cycle predominantly within the nucleus. Finally, mass spectrometric analysis demonstrated the position at Thr215 of NS1 protein is primary phosphorylation target site through Akt activation. The results together supported the functional importance of influenza virus NS1 with Akt, a core intra-cellular survival regulator

The dataset of proteins specifically interacted with activated TICAM-1

The presented data are related with our paper entitled “14-3-3-zeta participates in TLR3-mediated TICAM-1 signal-platform formation” (Funami et al., 2016) [1]. These data show the proteins which specifically bind to the activated (oligomerized) TICAM-1. Fifty-three proteins were identified as specifically interacted with oligomerized TICAM-1. Mutant TICAM-1 cannot form the active oligomer, so the proteins interacted with mutant TICAM-1 are dispensable for TICAM-1-signaling. Among 53 proteins, 14-3-3-zeta specifically interacts with oligomerized TICAM-1 to corroborate TICAM-1 signalosome. Keywords: TLR3, TICAM-1 (TRIF), 14-3-3, Signalosome, Proteome analysi