Identification of two novel BRCA1-partner genes in the DNA double-strand break repair pathway.

M1775R and A1789T are two missense variants located within the BRCT domains of BRCA1 gene. The M1775R is a known deleterious variant, while the A1789T is an unclassified variant that has been analyzed and classified as probably deleterious for the first time by our group. In a previous study, we described the expression profile of HeLa G1 cells transfected with the two variants and we found that they altered molecular mechanisms critical for cell proliferation and genome integrity. Considering that the mutations in the BRCA1 C terminus (BRCT) domains are associated to a phenotype with an altered ability in the DNA double-strand break repair, we chose three of the genes previously identified, EEF1E1, MRE11A, and OBFC2B, to be tested for an homologous recombination (HR) in vitro assay. For our purpose, we performed a gene expression knockdown by siRNA transfection in HeLa cells, containing an integrated recombination substrate (hprtDRGFP), for each of the target genes included BRCA1. The knockdown of BRCA1, OBFC2B, MRE11A, and EEF1E1 reduces the HR rate, respectively, of 97.6, 28.6, 41.8, and 42.3 % compared to cells transfected with a scrambled negative control duplex and all these differences are statistically significant (P < 0.05; Kruskal-Wallis test). Finally, we analyzed the effect of target gene depletion both on HR that on cell survival after DNA-damage induction by ionizing radiation. The clonogenic assay showed that the down-regulation of the target genes reduced the cell survival, but the effect on the HR, is not evident. Only the BRCA1-siRNA confirmed the inhibition effect on HR. Overall these results confirmed the involvement of MRE11A in the HR pathway and identified two new genes, OBFC2B and EEF1E1, which according to these data and the knowledge obtained from literature, might be involved in BRCA1-pathway.

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