Development of a Novel System to Capture DNA-associated Proteins



 DESCRIPTION (provided by applicant): In this application, we present a novel method for the identification, of proteins that associate with specific DNA loci. We refer to this technology as DNA-PCap (DNA-associated Protein Capture). The new technology enables the purification of proteins in the vicinity of specific genomic loci. This biochemical approach is based on recent technology described by the Roux laboratory that allows identification of proteins that are in close proximity to a "bait" based on a promiscuous biotin ligase (BirA*). In our approach as bait, we use an RNA- guided DNA binding protein (dCas9) to direct BirA* to specific DNA sequences. We validated this approach at telomeres, nucleo-protein complexes that protect chromosome ends. In this proposal, we will use DNA-PCap to identify proteins that bind to and suppress the activation of the Long Interspersed Elements (LINE-1), active transposable elements. In addition, we will apply DNA-PCap to identify transcriptional regulators of the catalytic subunit of telomerase (hTERT). This enzyme is silenced in human somatic cells, but it is re-activated in ~90% of human cancers. The precise mechanism of hTERT reactivation is currently not known. Isolation of factors that bind to the hTERT promoter has the potential to elucidate the mechanism of transcriptional regulation of this critical enzyme. Completion of this proposal will validate DNA-PCap as a universally applicable technique that allows the isolation of factors that bind to specific DNA elements. PUBLIC HEALTH RELEVANC: We developed a novel technique for the identification of proteins that are bound to specific DNA sequences. We termed this technique DNA-PCap (DNA-associated Protein Capture). We propose to apply DNA-PCap to identify factors that associate with repetitive elements and to the telomerase promoter.