TopBP1 interacts with BLM to maintain genome stability but is dispensable for preventing BLM degradation.

The Bloom syndrome helicase BLM and topoisomerase-IIβ-binding protein 1 (TopBP1) are key regulators of genome stability. It was recently proposed that BLM phosphorylation on Ser338 mediates its interaction with TopBP1, to protect BLM from ubiquitylation and degradation (Wang et al., 2013). Here, we show that the BLM-TopBP1 interaction does not involve Ser338 but instead requires BLM phosphorylation on Ser304. Furthermore, we establish that disrupting this interaction does not markedly affect BLM stability. However, BLM-TopBP1 binding is important for maintaining genome integrity, because in its absence cells display increased sister chromatid exchanges, replication origin firing and chromosomal aberrations. Therefore, the BLM-TopBP1 interaction maintains genome stability not by controlling BLM protein levels, but via another as-yet undetermined mechanism. Finally, we identify critical residues that mediate interactions between TopBP1 and MDC1, and between BLM and TOP3A/RMI1/RMI2. Taken together, our findings provide molecular insights into a key tumor suppressor and genome stability network.293FT cells, E1A antibody, and hr703 virus were gifts from Roger Grand, and DT40 cells and human LCLs were gifts from Julian Sale and Ian Hickson, respectively. We thank Nathan Ellis, Thanos Halazonetis, Frank Hänel, and Minoru Takata for plasmids; Grant Stewart and Yi Wang for antibodies; and Gabriel Balmus, Josep Forment, Abderrahmane Kaidi, Christine Schmidt, and Jon Travers for critical reading of the manuscript. This work was funded by a Worldwide Cancer Research International Fellowship and a WIMM/Medical Research Council Senior Non-Clinical Fellowship (MRCG0902418) to W.N., and by Polish Ministry of Science and Higher Education fellowship and Polish National Science Center grant number N303 571539 to J.N. The Jackson lab is funded by Cancer Research UK (CRUK) program grant C6/A11224, the European Research Council, and the European Community Seventh Framework Programme grant agreement number HEALTH-F2-2010-259893 (DDResponse). Core infrastructure funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK.This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.molcel.2015.02.01

Varied effect of fortification of kale sprouts with novel organic selenium compounds on the synthesis of sulphur and phenolic compounds in relation to cytotoxic, antioxidant and anti-inflammatory activity

Selenium deficiency in daily diet is a common problem in many countries, thus searching for new dietary sources of this trace element is an important scientific challenge. Selenium biofortified sprouts from Brassicaceae family are good candidates for new dietary selenium source, as they reveal one of the highest capability to synthesize and accumulate this element. As a part of this extensive search, the influence of novel selenium organic compounds on fortification of kale sprouts biological activity was investigated. The present study is focused on the evaluation of the influence of these compounds on the synthesis of glucosinolates, isothiocyanates, indoles and phenolic acids in kale sprouts, together with the determination of their impact on antioxidant, anti-inflammatory and cytotoxic activity on gastrointestinal, prostate, and thyroid normal and cancer cells. The present study yields the conclusion that fortification of kale sprouts with selenium organic compounds bearing benzoselenoate scaffold influences the production of isothiocyanates, phenolic acids, and enhances the antioxidant properties of fortified sprouts. Notably, fortification with compounds based on benzoselenoate scaffold display chemoprotective properties in various cancer types (gastric, thyroid, and prostate cancer). The present study can facilitate the design of future agrochemicals. Compounds bearing benzoselenoate scaffold or selenyl phenylpiperazine motif seem to be particularly promising for these purposes.This study was partly supported by Polish grants N42/DBS/000231 and N42/DBS/000167. The publication was created with the use of equipment’s (Biotek Synergy microplate reader and Dionex HPLC system) co-financed by the qLIFE Priority Research Area under the program “Excellence Initiative—Research University” (No. 06/IDUB/2019/94) at Jagiellonian University

Structural basis for the RING catalyzed synthesis of K63 linked ubiquitin chains

This work was supported by grants from Cancer Research UK (C434/A13067), the Wellcome Trust (098391/Z/12/Z) and Biotechnology and Biological Sciences Research Council (BB/J016004/1).The RING E3 ligase catalysed formation of lysine 63 linked ubiquitin chains by the Ube2V2–Ubc13 E2 complex is required for many important biological processes. Here we report the structure of the RING domain dimer of rat RNF4 in complex with a human Ubc13~Ub conjugate and Ube2V2. The structure has captured Ube2V2 bound to the acceptor (priming) ubiquitin with Lys63 in a position that could lead to attack on the linkage between the donor (second) ubiquitin and Ubc13 that is held in the active “folded back” conformation by the RING domain of RNF4. The interfaces identified in the structure were verified by in vitro ubiquitination assays of site directed mutants. This represents the first view of the synthesis of Lys63 linked ubiquitin chains in which both substrate ubiquitin and ubiquitin-loaded E2 are juxtaposed to allow E3 ligase mediated catalysis.PostprintPeer reviewe

Systematic characterization of deubiquitylating enzymes for roles in maintaining genome integrity.

DNA double-strand breaks (DSBs) are perhaps the most toxic of all DNA lesions, with defects in the DNA-damage response to DSBs being associated with various human diseases. Although it is known that DSB repair pathways are tightly regulated by ubiquitylation, we do not yet have a comprehensive understanding of how deubiquitylating enzymes (DUBs) function in DSB responses. Here, by carrying out a multidimensional screening strategy for human DUBs, we identify several with hitherto unknown links to DSB repair, the G2/M DNA-damage checkpoint and genome-integrity maintenance. Phylogenetic analyses reveal functional clustering within certain DUB subgroups, suggesting evolutionally conserved functions and/or related modes of action. Furthermore, we establish that the DUB UCHL5 regulates DSB resection and repair by homologous recombination through protecting its interactor, NFRKB, from degradation. Collectively, our findings extend the list of DUBs promoting the maintenance of genome integrity, and highlight their potential as therapeutic targets for cancer.This is the author's accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ncb302

Loss of ubiquitin E2 Ube2w rescues hypersensitivity of Rnf4 mutant cells to DNA damage

SUMO and ubiquitin play important roles in the response of cells to DNA damage. These pathways are linked by the SUMO Targeted ubiquitin Ligase Rnf4 that catalyses transfer of ubiquitin from a ubiquitin loaded E2 conjugating enzyme to a polySUMO modified substrate. Rnf4 can functionally interact with multiple E2s, including Ube2w, in vitro. Chicken cells lacking Rnf4 are hypersensitive to hyroxyurea, DNA alkylating drugs and DNA crosslinking agents, but this sensitivity is suppressed by simultaneous depletion of Ube2w. Cells depleted of Ube2w alone are not hypersensitive to the same DNA damaging agents. Similar results were also obtained in human cells. These data indicate that Ube2w does not have an essential role in the DNA damage response, but is deleterious in the absence of Rnf4. Thus, although Rnf4 and Ube2w functionally interact in vitro, our genetic experiments indicate that in response to DNA damage Ube2w and Rnf4 function in distinct pathways

Slx8 removes Pli1-dependent protein-SUMO conjugates including SUMOylated Topoisomerase I to promote genome stability

Peer reviewedPublisher PD

Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks

During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair. © 2014 Nakajima et al

Expression of arf tumor suppressor in spermatogonia facilitates meiotic progression in male germ cells

The mammalian Cdkn2a (Ink4a-Arf) locus encodes two tumor suppressor proteins (p16Ink4a and p19Arf) that respectively enforce the anti-proliferative functions of the retinoblastoma protein (Rb) and the p53 transcription factor in response to oncogenic stress. Although p19Arf is not normally detected in tissues of young adult mice, a notable exception occurs in the male germ line, where Arf is expressed in spermatogonia, but not in meiotic spermatocytes arising from them. Unlike other contexts in which the induction of Arf potently inhibits cell proliferation, expression of p19Arf in spermatogonia does not interfere with mitotic cell division. Instead, inactivation of Arf triggers germ cell-autonomous, p53-dependent apoptosis of primary spermatocytes in late meiotic prophase, resulting in reduced sperm production. Arf deficiency also causes premature, elevated, and persistent accumulation of the phosphorylated histone variant H2AX, reduces numbers of chromosome-associated complexes of Rad51 and Dmc1 recombinases during meiotic prophase, and yields incompletely synapsed autosomes during pachynema. Inactivation of Ink4a increases the fraction of spermatogonia in S-phase and restores sperm numbers in Ink4a-Arf doubly deficient mice but does not abrogate γ-H2AX accumulation in spermatocytes or p53-dependent apoptosis resulting from Arf inactivation. Thus, as opposed to its canonical role as a tumor suppressor in inducing p53-dependent senescence or apoptosis, Arf expression in spermatogonia instead initiates a salutary feed-forward program that prevents p53-dependent apoptosis, contributing to the survival of meiotic male germ cells

Targeting of SUMO substrates to a Cdc48-Ufd1-Npl4 segregase and STUbL pathway in fission yeast

In eukaryotes, the conjugation of proteins to the small ubiquitin-like modifier (SUMO) regulates numerous cellular functions. A proportion of SUMO conjugates are targeted for degradation by SUMO-targeted ubiquitin ligases (STUbLs) and it has been proposed that the ubiquitin-selective chaperone Cdc48/p97-Ufd1-Npl4 facilitates this process. However, the extent to which the two pathways overlap, and how substrates are selected, remains unknown. Here we address these questions in fission yeast through proteome-wide analyses of SUMO modification sites. We identify over a thousand sumoylated lysines in a total of 468 proteins and quantify changes occurring in the SUMO modification status when the STUbL or Ufd1 pathways are compromised by mutations. The data suggest the coordinated processing of several classes of SUMO conjugates, many dynamically associated with centromeres or telomeres. They provide new insights into subnuclear organization and chromosome biology, and, altogether, constitute an extensive resource for the molecular characterization of SUMO function and dynamics