Characterizing S. cerevisiae Pif1 residues: Identifying the nuclear localization signal and testing in vivo functions of residues associated with G-quadruplex DNA

Abstract

Maintaining genome integrity is a critical function of cells. Pif1, a multifunctional DNA helicase found in both the nucleus and mitochondria, promotes genome integrity. In this two-part study, mutational analysis was conducted to identify residues that are essential for Pif1 functions. In the first part, the previously undiscovered nuclear localization signal (NLS) of Pif1 was identified. Residues 781-Lys-Lys-Arg-Lys-784 were predicted to be the NLS through computational analysis and literature review. Cells with the pif1-781-784∆ allele displayed disrupted nuclear Pif1 functions, including Okazaki fragment (OF) maturation and telomerase inhibition, but preserved mitochondrial DNA (mtDNA) maintenance function. The functional NLS was then confirmed to be residues 781-784, because the phenotype of pif1-781-784∆ was rescued by the addition of the SV40 NLS sequence. The pif1-781-784∆ allele is a valuable experimental tool that allows researchers to explore the effects of the absence of Pif1 in the nucleus. In the second part of this study, the unwinding of G-quadruplex (G4) DNA by Pif1 was investigated in vivo. G4 DNA are secondary structures that result from noncanonical base-pairing, and a recent in vitro study identified mutations that reduce the ability of Pif1 to bind and unwind G4 DNA. The effects of these mutations on in vivo Pif1 functions were tested and showed that they disrupt OF maturation, but not mtDNA maintenance. As G4 structures impede fork progression and induce damage, elucidating the mechanisms of G4 unwinding by Pif1 is important to understanding the promotion of genome integrity.