PI 3 kinase related kinases-independent proteolysis of BRCA1 regulates Rad51 recruitment during genotoxic stress in human cells

• Published in: PloS one Vol. 5; no. 11; p. e14027
• Main Authors: Hammond-Martel, Ian, Pak, Helen, Yu, Helen, Rouget, Raphael, Horwitz, Andrew A, Parvin, Jeffrey D, Drobetsky, Elliot A, Affar, El Bachir
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 17.11.2010; Public Library of Science (PLoS)
• Subjects: Adducts; Alkylation; Analysis; Antineoplastic Agents, Alkylating - pharmacology; Apoptosis; Apoptosis - drug effects; Apoptosis - radiation effects; Ataxia; Ataxia Telangiectasia Mutated Proteins; Background radiation; Biochemistry/ and Repair; BRCA1 protein; BRCA1 Protein - metabolism; Breast cancer; Cell Biology/Cell Signaling; Cell Biology/Nuclear Structure and Function; Cell cycle; Cell Cycle - drug effects; Cell Cycle - radiation effects; Cell Cycle Proteins - metabolism; Cell Line, Tumor; Cells (Biology); Cells, Cultured; Chromatin; Chromatin - metabolism; Critical components; Deoxyribonucleic acid; Dismantling; DNA; DNA adducts; DNA Damage; DNA repair; DNA-Activated Protein Kinase - metabolism; DNA-Binding Proteins - metabolism; Down-Regulation - drug effects; Down-Regulation - radiation effects; Drug dosages; Genes; Genotoxicity; HCT116 Cells; HEK293 Cells; HeLa Cells; Homologous recombination; Homology; Humans; Immunoblotting; Inhibition; Ionizing radiation; Kinases; Localization; Male; Medicine; Methyl Methanesulfonate - pharmacology; Molecular Biology/DNA Repair; Mutagens; Mutation; Ovarian cancer; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation; Phosphotransferases; Proteasomes; Protein-Serine-Threonine Kinases - metabolism; Proteins; Proteolysis; Rad51 Recombinase - metabolism; Rad52 protein; Radiation; Radiation damage; Radiation effects; Recruitment; Signal transduction; Signaling; Stress (Physiology); Studies; Transcription factors; Tumor suppressor genes; Tumor Suppressor Proteins - metabolism; Ubiquitin-Protein Ligases - metabolism; Ultraviolet Rays; Womens health; San Francisco California; California; United States > US; Massachusetts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0014027

The function of BRCA1 in response to ionizing radiation, which directly generates DNA double strand breaks, has been extensively characterized. However previous investigations have produced conflicting data on mutagens that initially induce other classes of DNA adducts. Because of the fundamental and clinical importance of understanding BRCA1 function, we sought to rigorously evaluate the role of this tumor suppressor in response to diverse forms of genotoxic stress. We investigated BRCA1 stability and localization in various human cells treated with model mutagens that trigger different DNA damage signaling pathways. We established that, unlike ionizing radiation, either UVC or methylmethanesulfonate (MMS) (generating bulky DNA adducts or alkylated bases respectively) induces a transient downregulation of BRCA1 protein which is neither prevented nor enhanced by inhibition of PIKKs. Moreover, we found that the proteasome mediates early degradation of BRCA1, BARD1, BACH1, and Rad52 implying that critical components of the homologous recombination machinery need to be functionally abrogated as part of the early response to UV or MMS. Significantly, we found that inhibition of BRCA1/BARD1 downregulation is accompanied by the unscheduled recruitment of both proteins to chromatin along with Rad51. Consistently, treatment of cells with MMS engendered complete disassembly of Rad51 from pre-formed ionizing radiation-induced foci. Following the initial phase of BRCA1/BARD1 downregulation, we found that the recovery of these proteins in foci coincides with the formation of RPA and Rad51 foci. This indicates that homologous recombination is reactivated at later stage of the cellular response to MMS, most likely to repair DSBs generated by replication blocks. Taken together our results demonstrate that (i) the stabilities of BRCA1/BARD1 complexes are regulated in a mutagen-specific manner, and (ii) indicate the existence of mechanisms that may be required to prevent the simultaneous recruitment of conflicting signaling pathways to sites of DNA damage.