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Nucleic Acids Research, 2000, Vol. 28, No. 18 3478-3485
© 2000 Oxford University Press

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

John M. Cho, Daniel J. Song, Josee Bergeron, Naciba Benlimame, Marc S. Wold1 and Moulay A. Alaoui-Jamali*

Departments of Medicine, Oncology and Pharmacology, Lady Davis Institute for Medical Research of the Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada and 1Department of Biochemistry, University of Iowa College of Medicine, 4403 Bowen Science Building, Iowa City, IA 52242-1109, USA

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.

* To whom correspondence should be addressed. Tel: +1 514 340 8222; Fax: +1 514 340 7576; Email: mdaj@musica.mcgill.ca


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