Mediator of DNA damage checkpoint protein-1 (MDC1) is a recently identified nuclear protein that participates in DNA-damage sensing and signaling. Here we report that knockdown of MDC1 by RNA interference results in cellular hypersensitivity to the DNA cross-linking agent mitomycin C and ionizing radiation and in impaired homology-mediated repair of double-strand breaks in DNA. MDC1 forms a complex with Rad51 through a direct interaction with the forkhead-associated domain of MDC1, not the BRCA1 C-terminal domain. Depletion of MDC1 results in impaired formation of Rad51 ionizing radiation-induced foci, reduced amounts of nuclear and chromatin-bound Rad51, and a corresponding increase in Rad51 protein degradation. Together, our findings suggest that MDC1 functions in Rad51-mediated homologous recombination by retaining Rad51 in chromatin.

The DNA damage response pathway controlled by the BRCA and Fanconi Anemia (FA) genes can be disrupted by genetic or epigenetic mechanisms in breast cancer. Defects in this pathway may render the affected tumors hypersensitive to DNA damaging agents. The identification of these defects poses a challenge because of the large number of genes involved in the FA/BRCA pathway. Many pathway components form subnuclear repair protein foci upon exposure to ionizing radiation in-vitro, but it was unknown whether foci can be detected in live cancer tissues. Thus, the goal of this pilot study was to identify pathway defects by using a novel ex-vivo foci biomarker assay on tumor biopsies. Fresh pretreatment biopsy specimens from patients with locally advanced sporadic breast cancer were irradiated or mock-treated in the laboratory (ex-vivo). Foci formation of DNA repair proteins BRCA1, FANCD2, and RAD51 was detected by immunofluorescence microscopy. Three out of seven tumors showed intact radiation-induced foci formation while the other four tumors exhibited a defective foci response. Notably, three of the foci-defective tumors were ER/PR/HER2 (triple) negative, a phenotype that has been associated with BRCA1 deficiency. In conclusion, in this pilot study, we report the successful detection of BRCA1, FANCD2 and RAD51 foci in breast cancer biopsies irradiated exvivo. Our approach represents a potentially powerful biomarker assay for the detection of pre-existing and functionally important defects within the complex FA/BRCA pathway, which may ultimately allow us to tailor cancer treatment to the DNA repair profile of individual tumors.