A Temporal and Quantitative Proteomic Analysis of the MRN Complex in Response to DNA Damage

De Santis, Andrea Marie, Department of Biochemistry and Molecular Genetics, University of Virginia
Auble, David, Department of Biochemistry and Molecular Genetics, University of Virginia
Grant, Patrick, Department of Biochemistry and Molecular Genetics, University of Virginia
Templeton, Dennis, Department of Pathology, University of Virginia
Concannon, Patrick, Department of Public Health Sciences, University of Virginia

The MRN complex, composed of Mre11, Rad50, and Nbn, is essential, critically guarding against genomic instability. Its multifunctional role in the cellular response to DNA double-strand breaks begins the moment the complex senses and binds a broken piece of DNA. Rad50 is able to tether two pieces of DNA together while Mre11, with nuclease activity, provides the initial processing of the DNA broken ends that can then be recognized by downstream repair factors. Nbn is the only component of the complex that does not bind DNA directly. Instead, Nbn has phosphoprotein binding domains that enable it to interact with effector proteins. When the MRN complex binds a newly detected break in DNA, Nbn transiently interacts with the nuclear kinase ATM. This interaction is necessary for the proper activation kinetics of ATM as characterized by a delay in the phosphorylation of effector, repair, and checkpoint proteins. In particular, ATM phosphorylates serine 278 (S278) and S343 on Nbn. This phosphorylation event seems to be necessary for the proper activation of the intra-S-phase checkpoint, but it is not clear how the phosphorylation of S278 and S343 of Nbn translates into a DNA synthesis checkpoint. It is possible that other post-translational modifications may be involved. In order to investigate this biological event, a differential, temporal and quantitative proteomic analysis was designed where cells expressing Nbn wild-type protein were directly compared to cells expressing Nbn with serine-to-alanine mutations at S278 and S343 using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry. The nuclear protein, Tcof1, was identified as a novel binding iii partner of the MRN complex. Temporal protein SILAC ratios defined how Mre11 and Rad50 differentially associate with Nbn S278A/S343A and Nbn wt. Several ATMmediated phosphorylation events were temporally and differentially characterized. Events not mediated by ATM were also characterized and further investigated for biological significance. A serine-to-alanine mutant of a highly phosphorylated residue on Nbn, S432A, was lethal when expressed in NBS-ILB1 cells while Nbn S518A expressing cells were tetraploid, compared to Nbn wt expressing cells. Several novel aspects of Nbn and the MRN complex are defined that have implications on genomic instability and malignant transformations.

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PHD (Doctor of Philosophy)
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