Synthetic viability genomic screening defines Sae2 function in DNA repair.

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3' single-stranded DNA (ssDNA) generation by 5' DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2∆ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.We thank M.P. Longhese, R. Rothstein and J. Haber for providing strains and plasmids; Sir T. Blundell and T. Ochi for advice on structural biology and for providing comments to the manuscript. Research in the Jackson laboratory is funded by Cancer Research UK Programme Grant C6/A11224, the European Research Council and the European Community Seventh Framework Programme Grant Agreement No. HEALTH‐F2‐2010‐259893 (DDResponse). Core funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). SPJ receives his salary from the University of Cambridge, UK, supplemented by CRUK. TO, IG and FP were funded by Framework Programme Grant Agreement No. HEALTH‐F2‐2010‐259893 (DDResponse). FP also received funding from EMBO (Fellowship ALTF 1287‐2011); NG and IS are funded by the Wellcome Trust (101126/Z/13/Z). DJA and TMK were supported by Cancer Research UK and the Wellcome Trust (WT098051). PS and HN were supported by NIH grants RO1ES007061 and K99ES021441, respectively.This is the final version. It was first published by EMBO at http://emboj.embopress.org/content/early/2015/04/21/embj.201590973.lon

Synthetic viability genomic screening defines Sae2 function in DNA repair.

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3' single-stranded DNA (ssDNA) generation by 5' DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2∆ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.We thank M.P. Longhese, R. Rothstein and J. Haber for providing strains and plasmids; Sir T. Blundell and T. Ochi for advice on structural biology and for providing comments to the manuscript. Research in the Jackson laboratory is funded by Cancer Research UK Programme Grant C6/A11224, the European Research Council and the European Community Seventh Framework Programme Grant Agreement No. HEALTH‐F2‐2010‐259893 (DDResponse). Core funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). SPJ receives his salary from the University of Cambridge, UK, supplemented by CRUK. TO, IG and FP were funded by Framework Programme Grant Agreement No. HEALTH‐F2‐2010‐259893 (DDResponse). FP also received funding from EMBO (Fellowship ALTF 1287‐2011); NG and IS are funded by the Wellcome Trust (101126/Z/13/Z). DJA and TMK were supported by Cancer Research UK and the Wellcome Trust (WT098051). PS and HN were supported by NIH grants RO1ES007061 and K99ES021441, respectively.This is the final version. It was first published by EMBO at http://emboj.embopress.org/content/early/2015/04/21/embj.201590973.lon

Small-molecule-induced DNA damage identifies alternative DNA structures in human genes.

Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here we show that the G-quadruplex-interacting drug pyridostatin promotes growth arrest in human cancer cells by inducing replication- and transcription-dependent DNA damage. A chromatin immunoprecipitation sequencing analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein abundance and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target of this drug. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions

Swm1/Apc13 is an evolutionarily conserved subunit of the anaphase-promoting complex stabilizing the association of cdc16 and cdc27.

The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo

Elecsys® Total-Tau and Phospho-Tau (181P) CSF assays : Analytical performance of the novel, fully automated immunoassays for quantification of tau proteins in human cerebrospinal fluid

Background: Total tau (tTau) and phosphorylated 181P tau (pTau) are supportive diagnostic cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease. Manual CSF tau assays are limited by lot-to-lot and between-laboratory variability and long incubation/turnaround times. Elecsys® Total-Tau CSF and Phospho-Tau (181P) CSF immunoassays were developed for fully automated cobas e analyzers, allowing broader access in clinical practice and trials. Methods: Analytical performance, reproducibility, method comparisons with commercially available assays, and lot-to-lot and platform comparability (cobas e 601/411) of the Elecsys® CSF assays were assessed. Tau distributions and concentration ranges were evaluated in CSF samples from two clinical cohorts. Results: Both assays showed high sensitivity (limit of quantitation [LoQ]: 63 pg/mL [tTau]; 4 pg/mL [pTau]) and linearity over the measuring range (80–1300 pg/mL; 8–120 pg/mL), which covered the entire concentration range measured in clinical samples. Lot-to-lot and platform comparability demonstrated good consistency (Pearson's r: 0.998; 1.000). Multicenter evaluation coefficients of variation (CVs): repeatability, < 1.8%; intermediate precision, < 2.8%; between-laboratory variability, < 2.7% (both assays); and total reproducibility, < 6.7% (tTau) and < 4.7% (pTau). Elecsys® CSF assays demonstrated good correlation with commercially available tau assays. Conclusions: Elecsys® Total-Tau CSF and Phospho-Tau (181P) CSF assays demonstrate good analytical performance with clinically relevant measuring ranges; data support their use in clinical trials and practice