PI 3 Kinase Related Kinases-Independent Proteolysis of BRCA1 Regulates Rad51 Recruitment during Genotoxic Stress in Human Cells

Ian Hammond-Martel,Andrew A Horwitz,Raphael Rouget,Helen Pak,Jeffrey D Parvin,Helen Yu,Elliot A Drobetsky,El Bachir Affar,Art J Lustig

doi:10.1371/journal.pone.0014027

Abstract

BackgroundThe 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.Methodology/Principal FindingsWe 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 recombinaion 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.Conclusion/SignificanceTaken 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.

Highlights

Germline mutations in BRCA1 cause extremely high predisposition to breast and ovarian cancers
Conclusion/Significance: Taken together our results demonstrate that (i) the stabilities of BRCA1/BRCA1-associated RING domain 1 (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
Using an antibody recognizing the N-terminal region of BRCA1, we found that UVC induced a substantial decrease in levels of this protein at 3 hrs post-treatment, which became more marked by 6 hrs (Fig. 1A, top panel)

Summary

Introduction

Germline mutations in BRCA1 cause extremely high predisposition to breast and ovarian cancers. Through the BRCT domain, BRCA1/BARD1 interacts with either Abraxas, BACH1, or CtIP, along with other distinct cofactors, to form multiprotein complexes termed A, B, and C, respectively These complexes play important roles in the DNA damage response by exerting specific overlapping functions in cell cycle arrest and DNA repair [2,8]. One study reported that this protein accumulates in nuclear foci following treatment with methylmethanesulfonate (MMS) [30], whereas another showed that BRCA1 is downregulated by methyl methanethiosulfonate [31] It is not yet clear how BRCA1/BARD1 stability and subcellular localization are regulated in response to diverse classes of DNA adducts, other than DSBs, which trigger unique though overlapping signaling pathways. Data is provided suggesting that DNA damage signaling pathways might need to be coordinated in order to forestall the untimely recruitment of potentially conflicting DNA damage responses

Results

Discussion

Materials and Methods