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The MRE11, RAD50, and NBS1 genes encode proteins of the MRE11-RAD50-NBS1 (MRN) complex critical for proper maintenance of genomic integrity and tumour suppression; however, the extent and impact of their cancer-predisposing defects, and potential clinical value remain to be determined. Here, we report that among a large series of approximately 1000 breast carcinomas, around 3%, 7% and 10% tumours showed aberrantly reduced protein expression for RAD50, MRE11 and NBS1, respectively. Such defects were more frequent among the ER/PR/ERBB2 triple-negative and higher-grade tumours, among familial (especially BRCA1/BRCA2-associated) rather than sporadic cases, and the NBS1 defects correlated with shorter patients' survival. The BRCA1-associated and ER/PR/ERBB2 triple-negative tumours also showed high incidence of constitutively active DNA damage signalling (gammaH2AX) and p53 aberrations. Sequencing the RAD50, MRE11 and NBS1 genes of 8 patients from non-BRCA1/2 breast cancer families whose tumours showed concomitant reduction/loss of all three MRN-complex proteins revealed two germline mutations in MRE11: a missense mutation R202G and a truncating mutation R633STOP (R633X). Gene transfer and protein analysis of cell culture models with mutant MRE11 implicated various destabilization patterns among the MRN complex proteins including NBS1, the abundance of which was restored by re-expression of wild-type MRE11. We propose that germline mutations qualify MRE11 as a novel candidate breast cancer susceptibility gene in a subset of non-BRCA1/2 families. Our data have implications for the concept of the DNA damage response as an intrinsic anti-cancer barrier, various components of which become inactivated during cancer progression and also represent the bulk of breast cancer susceptibility genes discovered to date.

Homologous recombinational repair (HRR) of DNA damage is critical for maintaining genome stability and tumor suppression. RAD51 and BRCA2 colocalization in nuclear foci is a hallmark of HRR. BRCA2 has important roles in RAD51 focus formation and HRR of DNA double-strand breaks (DSBs). We previously reported that BCCIPalpha interacts with BRCA2. We show that a second isoform, BCCIPbeta, also interacts with BRCA2 and that this interaction occurs in a region shared by BCCIPalpha and BCCIPbeta. We further show that chromatin-bound BRCA2 colocalizes with BCCIP nuclear foci and that most radiation-induced RAD51 foci colocalize with BCCIP. Reducing BCCIPalpha by 90% or BCCIPbeta by 50% by RNA interference markedly reduces RAD51 and BRCA2 foci and reduces HRR of DSBs by 20- to 100-fold. Similarly, reducing BRCA2 by 50% reduces RAD51 and BCCIP foci. These data indicate that BCCIP is critical for BRCA2- and RAD51-dependent responses to DNA damage and HRR.

Background and Purpose: We previously determined that the density of a rapidly migrating DNA end-binding complex (termed 'band-A') predicts radiosensitivity of human normal and tumor cells. The goal of this study was first to identify the protein components of band-A and to determine if the protein levels of band-A components would correlate with band-A density and radiosensitivity., Patients and Methods: DNA end-binding protein complex (DNA-EBC) protein components were identified by adding antibodies specific for a variety of DNA repair-associated proteins to the DNA-EBC reaction and then noting which antibodies super-shifted various DNA-EBC bands. Band-A levels were correlated with SF2 for a panel of primary human fibroblasts heterozygous for sequence-proven mutations in BRCA1 or BRCA2. The nuclear protein levels of band-A components were determined in each BRCA1 heterozygote by western hybridization., Results: DNA-EBC analysis of human nuclear proteins revealed 10 identifiable bands. The density of the most rapidly migrating DNA-EBC band correlated closely with both BRCA-mutation status and radiosensitivity (r(2)=0.85). This band was absent in cells with homozygous mutations in their ataxia-telangiectasia-mutated protein (ATM) genes. This band was also completely supershifted by the addition of antibodies to ATM, Ku70, DNA ligase III, Rpa32, Rpa14, DNA ligase IV, XRCC4, WRN, BLM, RAD51 and p53. However, the intranuclear concentrations of these proteins did not correlate with either the SF2 or DNA-EBC density. Neither BRCA1 or BRCA2 could be detected in band-A., Conclusions: DNA-EBC analysis of human nuclear extracts resulted in 10 bands, at least six of which contained ATM. The density of one of the DNA-EBCs predicted the radiosensitization caused by BRCA haploinsufficiency, and this band contains Ku70, ATM, DNA ligase III, Rpa32, Rpa14, DNA ligase IV, XRCC4, WRN, BLM, RAD51 and p53 but not BRCA 1 or 2. The density of band-A was independent of the nuclear concentration of any of its known component.

Adriamycin and other DNA-damaging agents have been shown to reduce BRCA2 mRNA levels in breast cancer cell lines, but the mechanism by which this occurs is unknown. In this study, we show that adriamycin and mitomycin C, but not other DNA-damaging agents, repress BRCA2 promoter activity in a dose- and time-dependent manner. We demonstrate that the effect is dependent on wild type p53 and that adriamycin and p53 mediate repression of the BRCA2 promoter by inhibiting binding of an upstream stimulatory factor protein complex to the promoter. In addition, we present evidence indicating that adriamycin and other DNA-damaging agents reduce BRCA2 mRNA and protein levels by altering both BRCA2 mRNA stability and protein stability. Thus, BRCA2 levels in the cell are regulated by three independent mechanisms in a p53-dependent manner.