title: PI 3 kinase related kinases-independent proteolysis of BRCA1 regulates Rad51 recruitment during genotoxic stress in human cells.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medicine, Maisonneuve-Rosemont Hospital Research Center, University of Montréal, Montréal, Québec, Canada.
authors.forename: Ian
authors.initials: I
authors.lastname: Hammond-Martel
authors.forename: Helen
authors.initials: H
authors.lastname: Pak
authors.forename: Helen
authors.initials: H
authors.lastname: Yu
authors.forename: Raphael
authors.initials: R
authors.lastname: Rouget
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Horwitz
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Parvin
authors.forename: Elliot A
authors.initials: EA
authors.lastname: Drobetsky
authors.forename: El Bachir
authors.initials: el B
authors.lastname: Affar
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D064007:Ataxia Telangiectasia Mutated Proteins; D019313:BRCA1 Protein / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000528:radiation effects; D045325:HCT116 Cells; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008297:Male; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051135:Rad51 Recombinase / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D018906:Antineoplastic Agents, Alkylating; D064007:Ataxia Telangiectasia Mutated Proteins; D019313:BRCA1 Protein; D018797:Cell Cycle Proteins; D002843:Chromatin; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D008741:Methyl Methanesulfonate; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D051135:Rad51 Recombinase; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; C102449:BARD1 protein, human; C576324:ATM protein, human; C576597:ATR protein, human