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 replication in all eukaryotes. RPA participation in these pathways is mediated by single-stranded DNA binding and protein interactions. We herein identify 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 pull-down and co-immunoprecipitation. Fluorescence microscopy indicates that green fluorescence protein-tagged RBT1 is localized to the nucleus in vivo. RBT1 mRNA expression, determined by semi-quantitative RT-PCR, is significantly 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 normal 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 of significant variance between cell lines; the GAL4-RBT1 fusion protein has significantly higher transcriptional activity in human cancer cells compared to human normal primary non-immortalized epithelial cells. We propose that RBT1 is a novel transcriptional co-activator that interacts with RPA, and has significantly higher activity in transformed cells.
Cho
J M
JM
Departments of Medicine, Oncology and Pharmacology, Lady Davis Institute for Medical Research of the Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada.
Song
D J
DJ
Bergeron
J
J
Benlimame
N
N
Wold
M S
MS
Alaoui-Jamali
M A
MA
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D051716:Replication Protein A; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; C413405:SERTAD3 protein, human; D015534:Trans-Activators; D004247:DNA