Publications by Year: 2014

2014
Rajashree A Deshpande, Gareth J Williams, Oliver Limbo, Scott R Williams, Jeff Kuhnlein, Ji-hoon Lee, Scott Classen, Grant Guenther, Paul Russell, John A Tainer, and Tanya T Paull. 2AD. “ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling.” EMBO J, 33, 5, Pp. 482-500. Abstract
The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced 'closed' conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5' strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks.
Nodar Makharashvili, Anthony T Tubbs, Soo-Hyun Yang, Hailong Wang, Olivia Barton, Yi Zhou, Rajashree A Deshpande, Ji-hoon Lee, Markus Lobrich, Barry P Sleckman, Xiaohua Wu, and Tanya T Paull. 2AD. “Catalytic and noncatalytic roles of the CtIP endonuclease in double-strand break end resection.” Mol Cell, 54, 6, Pp. 1022-33. Abstract
The carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) is known to function in 5' strand resection during homologous recombination, similar to the budding yeast Sae2 protein, but its role in this process is unclear. Here, we characterize recombinant human CtIP and find that it exhibits 5' flap endonuclease activity on branched DNA structures, independent of the MRN complex. Phosphorylation of CtIP at known damage-dependent sites and other sites is essential for its catalytic activity, although the S327 and T847 phosphorylation sites are dispensable. A catalytic mutant of CtIP that is deficient in endonuclease activity exhibits wild-type levels of homologous recombination at restriction enzyme-generated breaks but is deficient in processing topoisomerase adducts and radiation-induced breaks in human cells, suggesting that the nuclease activity of CtIP is specifically required for the removal of DNA adducts at sites of DNA breaks.
Ji-hoon Lee, Zhi Guo, Logan R Myler, Suting Zheng, and Tanya T Paull. 2AD. “Direct activation of ATM by resveratrol under oxidizing conditions.” PLoS One, 9, 6, Pp. e97969. Abstract
Resveratrol has been widely reported to reduce cancer progression in model systems and to selectively induce cell death in transformed cell lines. Many enzymes have been reported to respond to resveratrol in mammalian cells, including the Ataxia-Telangiectasia Mutated (ATM) protein kinase that acts in DNA damage recognition, signaling, and repair. Here we investigate the responses of ATM to resveratrol exposure in normal and transformed human cell lines and find that ATM autophosphorylation and substrate phosphorylation is stimulated by resveratrol in a manner that is promoted by reactive oxygen species (ROS). We observe direct stimulatory effects of resveratrol on purified ATM in vitro and find that the catalytic efficiency of the kinase on a model substrate is increased by resveratrol. In the purified system we also observe a requirement for oxidation, as the effect of resveratrol on ATM signaling is substantially reduced by agents that prevent disulfide bond formation in ATM. These results demonstrate that resveratrol effects on ATM are direct, and suggest a mechanism by which the oxidizing environment of transformed cells promotes ATM activity and blocks cell proliferation.
Tanya T Paull and Rajashree A Deshpande. 2AD. “The Mre11/Rad50/Nbs1 complex: recent insights into catalytic activities and ATP-driven conformational changes.” Exp Cell Res, 329, 1, Pp. 139-47.
Qiong Fu, Julia Chow, Kara A Bernstein, Nodar Makharashvili, Sucheta Arora, Chia-Fang Lee, Maria D Person, Rodney Rothstein, and Tanya T Paull. 2AD. “Phosphorylation-regulated transitions in an oligomeric state control the activity of the Sae2 DNA repair enzyme.” Mol Cell Biol, 34, 5, Pp. 778-93. Abstract
In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterized in vivo in the budding yeast Saccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state.
Olivia Barton, Steffen C Naumann, Ronja Diemer-Biehs, Julia Künzel, Monika Steinlage, Sandro Conrad, Nodar Makharashvili, Jiadong Wang, Lin Feng, Bernard S Lopez, Tanya T Paull, Junjie Chen, Penny A Jeggo, and Markus Löbrich. 2AD. “Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1.” J Cell Biol, 206, 7, Pp. 877-94. Abstract
DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein-interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku(-/-) mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.
Yi Zhou, Pierre Caron, Gaëlle Legube, and Tanya T Paull. 2AD. “Quantitation of DNA double-strand break resection intermediates in human cells.” Nucleic Acids Res, 42, 3, Pp. e19. Abstract
5' strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. Here we develop a novel method to quantitatively measure single-stranded DNA intermediates in human cells and find that the 5' strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle-dependent manner. Depletion of CtIP, Mre11, Exo1 or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of Brca1 does not, suggesting that the role of Brca1 in HR is primarily to promote Rad51 filament formation, not to regulate end resection.
Tanya T Paull and Ji-hoon Lee. 2AD. “Rad17, the clamp loader that loads more than clamps.” EMBO J, 33, 8, Pp. 783-5. Abstract
Rad17 is best known as a checkpoint clamp loader in the activation of ATR kinase signaling. A new study in The EMBO Journal suggests that it also plays a role in initial recruitment of the MRN complex to sites of DNA double-strand breaks, thereby promoting early ATM checkpoint responses and homologous recombination repair.