Identification of proteins that specifically interact with triplex DNA structures: Role of heat shock proteins in cells undergoing triplex mediated gene modifications

Postdoc Assoc Ther Radiology
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Site specific gene modifications using triplex-forming oligonucleotides (TFOs) has been extensively used to induce recombination in numerous episomal and chromosomal DNA in different mammalian cells. Using TFOs and triplex-forming peptide nucleic acids (TFPs), a class of TFOs, our lab has been successful in stimulating recombination and site specific gene modifications of clinically significant mammalian genes such as the beta-globin, gamma-globin and CCR5 genes in both somatic and hematopoietic cells to create functional counterparts of these genes that alleviate the disease phenotype. Even though nucleotide excision repair (NER) factor Xeroderma pigmentosa A (XPA) is required for the TFO-mediated gene modifications, the mechanism by which the TFOs stimulate recombination is not well understood. Understanding the mechanistic details of the TFO-mediated gene modifications will not only enable us in improving the efficiency of correcting disease genes but will also help in understanding genome instability caused by naturally occurring triplex structures in chromosomal DNA that are considered to be hot spots for chromosomal rearrangements. Here we demonstrate the use of biotinylated DNA structures to identify proteins that bind specifically to the triplex DNA /in vitro/. Preliminary experiments indicate the recruitment of heat shock proteins to these triplex structures. Recent evidence from other groups, show the potential role of heat shock proteins and their chaperone machinery in DNA repair. In this study we report the status of heat shock proteins such as HSP90 and HSP70 in cells undergoing
TFO-mediated site specific gene modifications.