Publications by Year: 2007

2007
Shunichi Takeda, Kyoko Nakamura, Yoshihito Taniguchi, and Tanya T Paull. “Ctp1/CtIP and the MRN complex collaborate in the initial steps of homologous recombination.” Mol Cell, 28, 3, Pp. 351-2. Abstract
In a recent issue of Molecular Cell, Limbo et al. (2007) identify a new homologous recombination factor, Ctp1 in S. pombe, a homolog of Sae2/Com1 in S. cerevisiae and CtIP in mammals, that operates cooperatively with the MRN complex.
Clayton R Hunt, Raj K Pandita, Andrei Laszlo, Ryuji Higashikubo, Manjula Agarwal, Tetsuya Kitamura, Arun Gupta, Nicole Rief, Nobuo Horikoshi, Rajeskaran Baskaran, Ji-hoon Lee, Markus Löbrich, Tanya T Paull, Joseph L Roti Roti, and Tej K Pandita. “Hyperthermia activates a subset of ataxia-telangiectasia mutated effectors independent of DNA strand breaks and heat shock protein 70 status.” Cancer Res, 67, 7, Pp. 3010-7. Abstract
All cells have intricately coupled sensing and signaling mechanisms that regulate the cellular outcome following exposure to genotoxic agents such as ionizing radiation (IR). In the IR-induced signaling pathway, specific protein events, such as ataxia-telangiectasia mutated protein (ATM) activation and histone H2AX phosphorylation (gamma-H2AX), are mechanistically well characterized. How these mechanisms can be altered, especially by clinically relevant agents, is not clear. Here we show that hyperthermia, an effective radiosensitizer, can induce several steps associated with IR signaling in cells. Hyperthermia induces gamma-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced gamma-H2AX foci formation is dependent on ATM but independent of heat shock protein 70 expression. Hyperthermia also enhanced ATM kinase activity and increased cellular ATM autophosphorylation. The hyperthermia-induced increase in ATM phosphorylation was independent of Mre11 function. Similar to IR, hyperthermia also induced MDC1 foci formation; however, it did not induce all of the characteristic signals associated with irradiation because formation of 53BP1 and SMC1 foci was not observed in heated cells but occurred in irradiated cells. Additionally, induction of chromosomal DNA strand breaks was observed in IR-exposed but not in heated cells. These results indicate that hyperthermia activates signaling pathways that overlap with those activated by IR-induced DNA damage. Moreover, prior activation of ATM or other components of the IR-induced signaling pathway by heat may interfere with the normal IR-induced signaling required for chromosomal DNA double-strand break repair, thus resulting in increased cellular radiosensitivity.
Venugopal Bhaskara, Aude Dupré, Bettina Lengsfeld, Ben B Hopkins, Angela Chan, Ji-hoon Lee, Xiaoming Zhang, Jean Gautier, Virginia Zakian, and Tanya T Paull. “Rad50 adenylate kinase activity regulates DNA tethering by Mre11/Rad50 complexes.” Mol Cell, 25, 5, Pp. 647-61. Abstract
Mre11 and Rad50 are the catalytic components of a highly conserved DNA repair complex that functions in many aspects of DNA metabolism involving double-strand breaks. The ATPase domains in Rad50 are related to the ABC transporter family of ATPases, previously shown to share structural similarities with adenylate kinases. Here we demonstrate that Mre11/Rad50 complexes from three organisms catalyze the reversible adenylate kinase reaction in vitro. Mutation of the conserved signature motif reduces the adenylate kinase activity of Rad50 but does not reduce ATP hydrolysis. This mutant resembles a rad50 null strain with respect to meiosis and telomere maintenance in S. cerevisiae, correlating adenylate kinase activity with in vivo functions. An adenylate kinase inhibitor blocks Mre11/Rad50-dependent DNA tethering in vitro and in cell-free extracts, indicating that adenylate kinase activity by Mre11/Rad50 promotes DNA-DNA associations. We propose a model for Rad50 that incorporates both ATPase and adenylate kinase reactions as critical activities that regulate Rad50 functions.
Bettina M Lengsfeld, Alison J Rattray, Venugopal Bhaskara, Rodolfo Ghirlando, and Tanya T Paull. “Sae2 is an endonuclease that processes hairpin DNA cooperatively with the Mre11/Rad50/Xrs2 complex.” Mol Cell, 28, 4, Pp. 638-51. Abstract
Mre11/Rad50 complexes in all organisms function in the repair of DNA double-strand breaks. In budding yeast, genetic evidence suggests that the Sae2 protein is essential for the processing of hairpin DNA intermediates and meiotic double-strand breaks by Mre11/Rad50 complexes, but the biochemical basis of this functional relationship is not known. Here we demonstrate that recombinant Sae2 binds DNA and exhibits endonuclease activity on single-stranded DNA independently of Mre11/Rad50 complexes, but hairpin DNA structures are cleaved cooperatively in the presence of Mre11/Rad50 or Mre11/Rad50/Xrs2. Hairpin structures are not processed at the tip by Sae2 but rather at single-stranded DNA regions adjacent to the hairpin. Truncation and missense mutants of Sae2 inactivate this endonuclease activity in vitro and fail to complement Deltasae2 strains in vivo for meiosis and recombination involving hairpin intermediates, suggesting that the catalytic activities of Sae2 are important for its biological functions.