Authors: Austin Meyer, Gavin Grimmett, Martina Gaspari
Mentors: Martina Gaspari
Insitution: Utah Tech University
DNA can form non-canonical base pairings, resulting in structures distinct from the conventional double helix. One such example is the Hoogsteen base pairing, which can give rise to triple helix DNA. Triple helix DNA plays essential roles in regulating DNA metabolism and gene function, but its unusual nature is inherently mutagenic, sometimes leading to conditions like cancer. Recent research has uncovered that colorectal tumors exhibit significantly higher activity of triplex DNA binding proteins compared to their normal tissue. To explore this correlation, this study aims to employ cell and molecular biology techniques to investigate the relationship between the rise in triple helix DNA binding proteins and the presence of DNA triple helices. We initiated our study by extracting total RNA from HeLa cells, followed by the design and synthesis of specific primers for cloning ORC2 and ORC4 by RT-PCR. The experiment is further designed to analyze the potential consequences of ORC2 and ORC4 overexpression in human cell lines. Expression vectors containing ORC2 and ORC4 will be transfected into WI-38 cell lines comparing them to untransfected ones with hope of observing an increase of the proteins of interest and of triple helix DNA. The cell lines will be lysed, the total protein will be extracted, and Western blot will be conducted for comparison. Triple helix DNA will also be measured by calorimetric melting and high-speed centrifugation.