Helicase Mechanism and G-Quadruplex Signaling

Project Summary The MIRA will support the productive, long running Research Program in the Raney lab. The mechanism and regulation of DNA helicase activities are being addressed in Project 1 whereas signaling mechanisms for G- quadruplexes follows in Project 2. Helicases are molecular motor proteins that transduce chemical energy from ATP hydrolysis into mechanical energy capable of unwinding dsDNA, translocating on ssDNA, unfolding quadruplex DNA and remodeling protein-DNA complexes by moving protein along DNA. Two percent of the yeast genome encodes helicases, and the human genome encodes over 100 helicases. Each metabolic pathway involving nucleic acids such as replication, transcription, repair, translation, and other processes requires the activity of one or more helicases. Many cancers are correlated with helicase mutations. Many viruses such as SARS2, ZIKA, West Nile, Ebola, and many others encode one or more helicases that is essential for viral replication, therefore helicases can be targets for-cancer and anti-viral development. The over-arching question for Project 1 is “what is the mechanism utilized by a helicase to perform multiple functions?” We are utilizing the yeast Pif1 helicase to address this question and others. Pif1 is in the largest superfamily of helicases. The biochemical and biological studies needed to determine the underlying mechanisms are facilitated in the yeast system. Pif unwinds duplex DNA, unfolds G-quadruplex DNA (G4DNA), translocates on single-stranded DNA, and remodels DNA bound to proteins by moving the proteins. Experiments here will address the hypothesis that these activities can be understood within a unified mechanism. A major gap is the lack of a consensus for how energy from ATP hydrolysis is transduced into molecular motion that can be used to unwind dsDNA or unfold quadruplex DNA. Another major gap includes the lack of knowledge of how helicases fit into macro-molecular machines such as the mitochondrial DNA replication holoenzyme. We have identified key amino acids that strongly control energy transduction through novel helicase motifs and we will determine the mechanism of these amino acids. Project 2 will address the over-arching question of “what is the function of the guanine-rich DNA sequences that can fold into G-quadruplex structures (G4DNA)?” These sequences clearly play important roles in replication, transcription, translation, and repair because they appear in highly disproportionate levels in the genome including promoters, telomeres, and mitochondrial DNA. In Project 2, we will determine whether G4DNA is excised from the genome through an excision repair mechanism. DNA repair mechanisms often remove damaged DNA from the genome. Our hypothesis is that G4DNA is excised, resulting in a signaling molecule that triggers a cellular response affecting many pathways including stress granule formation. We will test this hypothesis through a variety of structural, molecular, and cellular approaches. Initially, we will perform screens for nucleases that excise G4DNA. Projects 1 and 2 will continue to have a strong and lasting impact through the work in this Research Program.