TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their resolution.

During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIα (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.We thank Drs G. Stewart and F. Esashi for cell lines, Professor T.D. Halazonetis, Dr G.J. Gorbsky and Dr G. Stewart for plasmids and antibodies. We also thank Dr C. Lagerholm (Wolfson Imaging Centre, Oxford) and Dr D. Waithe (CBRG, Oxford) for their help with microscopy and image analysis, and the Mass Spectrometry Laboratory (IBB PAS) for their work on analyses of GFP–TOP2A immunoprecipitation experiments. We also thank Professor I. Hickson for helpful comments on the manuscript. This work was funded by a Worldwide Cancer Research International Fellowship (to W.N.), a WIMM/Medical Research Council Senior Non-Clinical Fellowship (to W.N.), a Polish Ministry of Science and Higher Education fellowship (to J.N.) and Polish National Science Center grant N N303 571539 (to J.N.).This is the final published version. It first appeared at http://www.nature.com/ncomms/2015/150312/ncomms7572/full/ncomms7572.html#abstract

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2.

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.Work in W.N.’s laboratory is funded by ICR Intramural Grant and Cancer Research UK Programme (A24881). R.A.S. and L.S. were supported by WIMM Senior Non-Clinical Fellowship awarded to W.N. M.M.S. and M.A.B. were supported by the Intramural Research Program of the NIH, National Institute on Aging, United States (Z01-AG000746-08). Work in S.G.’s laboratory is supported by BRFAA Intramural Funds. V.C. was supported by John S. Latsis Public Benefit Foundation and Alexander S. Onassis Public Benefit Foundation. Work in the P.P.’s laboratory is supported by grants from the Agence Nationale pour la Recherche (ANR), the Ligue Contre le Cancer (équipe labellisée), SIRIC Montpellier Cancer (INCa Inserm DGOS 12553), and the MSDAvenir fund

Chatpal Chatbot dialogue data set

The scripts used in the ChatPal chatbot are freely available as an output from the ChatPal project. The datasets contain the chatbot utterances in English, Swedish, Finnish and Scottish Gaelic. Any replies collected from users through the ChatPal chatbot are not included in these data. Datasets are available in csv format and contain Unicode character encodings (UTF-8). Disclaimer: The datasets are open access, should be used appropriately and can be repurposed. However, the ChatPal project team are not responsible for how you chose to use the data or repurpose the content

Could lymphatic mapping and sentinel node biopsy provide oncological providence for local resectional techniques for colon cancer? A review of the literature

<p>Abstract</p> <p>Background</p> <p>Endoscopic resectional techniques for colon cancer are undermined by their inability to determine lymph node status. This limits their application to only those lesions at the most minimal risk of lymphatic dissemination whereas their technical capacity could allow intraluminal or even transluminal address of larger lesions. Sentinel node biopsy may theoretically address this breach although the variability of its reported results for this disease is worrisome.</p> <p>Methods</p> <p>Medline, EMBASE and Cochrane databases were interrogated back to 1999 to identify all publications concerning lymphatic mapping for colon cancer with reference cross-checking for completeness. All reports were examined from the perspective of in vivo technique accuracy selectively in early stage disease (i.e. lesions potentially within the technical capacity of endoscopic resection).</p> <p>Results</p> <p>Fifty-two studies detailing the experiences of 3390 patients were identified. Considerable variation in patient characteristics as well as in surgical and histological quality assurances were however evident among the studies identified. In addition, considerable contamination of the studies by inclusion of rectal cancer without subgroup separation was frequent. Indeed such is the heterogeneity of the publications to date, formal meta-analysis to pool patient cohorts in order to definitively ascertain technique accuracy in those with T1 and/or T2 cancer is not possible. Although lymphatic mapping in early stage neoplasia alone has rarely been specifically studied, those studies that included examination of false negative rates identified high T3/4 patient proportions and larger tumor size as being important confounders. Under selected circumstances however the technique seems to perform sufficiently reliably to allow it prompt consideration of its use to tailor operative extent.</p> <p>Conclusion</p> <p>The specific question of whether sentinel node biopsy can augment the oncological propriety for endoscopic resective techniques (including Natural Orifice Transluminal Endoscopic Surgery [NOTES]) cannot be definitively answered at present. Study heterogeneity may account for the variability evident in the results from different centers. Enhanced capacity (perhaps to the level necessary to consider selective avoidance of en bloc mesenteric resection) by its confinement to only early stage disease is plausible although not proven. Specific study of the technique in early stage tumors is clearly essential before proffering this approach.</p

Protein-protein interactions, sub-cellular localisation and mobility of the human Cdc45 protein.

Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. The replication factor Cdc45 has essential functions in the initiation and elongation steps of eukaryotic DNA replication and plays a role in the intra-S-phase checkpoint. Its interactions with a variety of replication proteins forming two central complexes, Cdc45/Mcm2-7/GINS (CMG) the putative replicative helicase of eukaryotes, and replisome progression complex (RPC) during the cell cycle and after intra-S-phase checkpoint activation remain to be fully characterized. The C terminal part of Cdc45 is important for its interaction with Claspin. The interactions of human Cdc45 with the three replication factors Claspin, replication protein A (RPA) and DNA polymerase ¿ are maximal during S phase. Following UVC-mediated DNA damage, Cdc45-Claspin complex formation is reduced whereas the binding of Cdc45 to RPA is not affected. We also show that treatment of cells with UCN-01, Caffeine or Wortmannin does not rescue the UV-mediated destabilisation of Cdc45-Claspin interactions, suggesting that the loss of interaction between Cdc45 and Claspin occurs independently of ATR activation in the intra-S-phase checkpoint. The sub-cellular localisation of Cdc45 in the cell cycle and following activation of the intra-S-phase checkpoint was also determined. Cdc45 showed a specific nuclear and nucleolar localisation and the Cdc45 association with nucleoli was abolished following UV damage and inhibiting nucleolar transcription. We then determined the regions of Cdc45 needed for its localisation to nuclear compartments. Spcifically, aa101-155 and aa156-169 are required for recruitment of Cdc45 to the nucleus and nucleolus, respectively. Measuring the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS) in vivo in asynchronous cells and in cells synchronized at the G1/S transition and during S phase showed that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the in vivo FCS data. Analysis of the mobility of eGFP-Cdc45 and the size of complexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. These findings suggest that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damag

Regulation of cdc45 in the cell cycle and after dna damage

The Cdc (cell division cycle) 45 protein has a central role in the regulation of the initiation and elongation stages of eukaryotic chromosomal DNA replication. In addition, it is the main target for a Chk1 (checkpoint kinase i)-dependent Cdc25/CDK2 (cyclin-dependent kinase 2)-independent DNA damage checkpoint signal transduction pathway following low doses of 8PDE (benzo[a]pyrene dihydrodiol epoxide) treatment, which causes DNA damage similar to UV-induced adducts. cdc45 interacts physically and functionally with the putative eukaryotic replicative DNA helicase, the MCM (mini-chromosome maintenance) complex, and forms a helicase active \u27supercomplex\u27, the CMG [cdc45-MCM2-7-GINS (go-ichi-ni-san)] complex. These known protein-protein interactions, as well as unknown interactions and post-translational modifications, may be important for the regulation of Cdc45 and the initiation of DNA replication following DNA damage. Future studies will help to elucidate the molecular basis of this newly identified S-phase checkpoint pathway which has Cdc45 as a target