HDR assay assesses the ability of a VUS to GANT61 promote homologous recombination of double strand breaks. Currently only VUS located in the RING domain have been screened but this assay needs further validation. In addition, although the p.Arg71Gly variant was shown to be fully active in the HDR assay it is known to be pathogenic due to the production of an aberrant cryptic splicing site. This highlights the fact that not a single assay can determine functional abrogation of a VUS, as different mechanisms may be affected by different variants irrespective of their location. The Sharan group developed elegant mouse models for examining VUS for both BRCA1 and BRCA2. They engineered mouse embryonic stem cells with a knock-out allele and a conditional BRCA1 allele. Lethality can be rescued by incorporation of the wild type BRCA1. In this model variants that do not rescue lethality are considered pathogenic. The limitations of this model are that lethality of human BRCA1 is examined in mouse and not in human embryonic stem cells; it is time consuming and if a variant is not lethal then functional assays, as well as multifactorial probability-based GDC-0199 analysis still need to be performed for its classification. The major drawback of many of the functional assays currently being used is that they are not interrogating BRCA1 function in a holistic manner, as they are restricted in expressing a specific domain or assessing a single specific function. These limitations are imposed by the large size of the full-length BRCA1 protein. Therefore multiple functional assays in combination with multifactorial probability-based analysis are usually necessary to decipher the pathogenicity and mechanism of action of a given variant. Nevertheless domain specific assays are useful for assessing the function of a given variant, especially when no other tools are available. In this manuscript, in an attempt to overcome these limitations, we describe a model of assessing the function of fulllength BRCA1 protein in transiently transfected cells. This model can be adapted for screening multiple functions of BRCA1, overcoming the use of truncated forms of the protein which can lead to false observations. The proposed system enables the direct and simultaneous testing of the integrity of BRCA1 complexes, the sub-cellular localization of BRCA1 as well as the ability of BRCA1 to respond to genotoxic stress and migrate at sites of DSBs along with other interacting proteins of the DNA repair machinery. We hereby present the results obtained when this system was used to investigate the ability of p.Ser36Tyr BRCA1 novel VUS identified in 4 unrelated Cypriot families, to interact with BARD1, to promote the formation of ubiquitin chains following DNA damage and its sub-cellular localization. Immunofluorescence analysis of transfected S-phase synchronized cells further supported the above observations. In many cells ectopically expressing the p.Ser36Tyr BRCA1 variant, both BRCA1 and BARD1 were not entirely mobilized to the nucleus, as both proteins were also present in the cytoplasm. The retention of BRCA1 p.Ser36Tyr variant in the cytoplasm of transfected cells was more pronounced following genotoxic stress with HU in S-phase synchronized cells, thus explaining the failure of this variant to co-precipitate high levels of BARD1 in the nuclear extracts of damaged cells.