RBT1,a novel transcriptional co-activator, binds the second subunit of Replication Protein A

Replication Protein A (RPA) is required for DNA recombination, repair and r eplication in all eukaryotes, RPA participation in these pathways is mediat ed by single-stranded DNA binding and protein interactions. We herein ident ify a novel protein, Replication Protein Binding Trans-Activator (RBT1), in a yeast two-hybrid assay employing the second subunit of human RPA (RPA32) as bait. RBT1-RPA32 binding was confirmed by glutathione S-transferase pul l-down and co-immunoprecipitation. Fluorescence microscopy indicates that g reen fluorescence protein-tagged RBT1 is localized to the nucleus in vivo. RBT1 mRNA expression, determined by semi-quantitative RT-PCR, is significan tly higher in cancer cell lines MCF-7, ZR-75, SaOS-2 and H661, compared to the cell lines normal non-immortalized human mammary epithelial cells and n ormal non-immortalized human bronchial epithelial cells. Further, yeast and mammalian one-hybrid analysis shows that RBT1 is a strong transcriptional co-activator, Interestingly, mammalian transactivation data is indicative o f significant variance between cell lines; the GAL4-RBT1 fusion protein has significantly higher transcriptional activity in human-cancer cells compar ed to human normal primary non-immortalized epithelial cells. We propose th at RBT1 is a novel transcriptional co-activator that interacts with RPA, an d has significantly higher activity in transformed cells.