The findings were further supported by the immunofluorescence data as BRCA1 OTX015 nuclear foci co-localized with BARD1 foci, in S-phase synchronized cells and were mobilized into more concentrated foci after treatment with HU. Despite the nuclear localization of BRCA1 in S-phase synchronized cells, it was observed that in many cells BRCA1 was also located in the cytoplasm and this was more evident in the p.Ser36Tyr variant transfected cells. Cell fractionation into nuclear and cytosolic extracts, revealed that a proportion of the p.Ser36Tyr BRCA1:BARD1 complex remained within the cytoplasm and its cytoplasmic retention was more pronounced following genotoxic stress with HU. Within the RING domain of BRCA1 there are two nuclear export sequences that signal BRCA1 shuttling to the cytoplasm and downstream the RING domain there are two nuclear localization sequences. BRCA1 shuttling in and out of the nucleus is cell-cycle dependent; and its import to the nucleus can take place by two independent mechanisms: either through the importins-a/b which are dependent on GTP hydrolysis and binding of importin-a to the NLS of BRCA1, or through its interaction with BARD1, where the NLS of BARD1 mediates the translocation. Its nuclear export is mediated by the interaction of the NES of BRCA1 with the CRMI nuclear export receptor and this process is inhibited when BRCA1 is in complex with BARD1. It is plausible that the reduced ability of the p.Ser36Tyr variant to translocate into the nucleus might be due to an interference with one of the nuclear shuttling mechanisms. This mutation may cause conformational changes to the protein that mask the NLS binding site for importin-a, or the transport mechanism with BARD1 may be impaired, due to protein instability or weaker protein-protein interaction due to lower affinity, or conformational changes. The NES of BRCA1 is masked through its interaction with BARD1, thus blocking the export of BRCA1 from the nucleus. It is plausible that the p.Ser36Tyr change induces conformational changes that result in partial blocking of the NES, thus directing the heterodimer into the cytoplasm. Serine at position 36 is located in the b-sheet of the RING domain and it is likely that its substitution by tyrosine disrupts the structure and causes an overall conformational change. The heterodimer BRCA1:BARD1 exhibits E3 ligase activity through the RING domain of BRCA1 as it promotes the formation of polyubiquitin conjugates in collaboration with the UbcH5C E2 enzyme. Following genotoxic stress, BRCA1’s ligase activity is further enhanced by SUMO modification. The E3 ligase activity of the heterodimer has been implicated in DNA WZ8040 in vivo damage repair by homologous recombination during the S-phase of the cell cycle. Indeed it has been shown that foci of BRCA1, conjugated ubiquitin and cH2AX co-localize in the nucleus of S-phase synchronized cells, following genotoxic stress. The p.Ser36Tyr BRCA1 variant also demonstrated a reduced ability to promote formation of conjugated ubiquitin foci at sites of DNA damage. Compared to wild type BRCA1, this variant failed in most cases to co-localize with conjugated ubiquitin chains, and this failure was even more pronounced than that of the known pathogenic variant p.Cys61Gly, which appears to have some residual activity as it can co-localize with BARD1, conjugated ubiquitin foci and Rad51, in agreement with the findings of Drost and colleagues.