Insights into the structural and mechanistic basis of multifunctional S. cerevisiae Pif1p helicase

Qingman

Journal of Biological Chemistry

et al. 2018

Self Cite

Edited by Patrick Sung G-quadruplexes (G4s) are four-stranded DNA structures formed by Hoogsteen base pairing between stacked sets of four guanines. Pif1 helicase plays critical roles in suppressing genomic instability in the yeast Saccharomyces cerevisiae by resolving G4s. However, the structural properties of G4s in S. cerevisiae and the substrate preference of Pif1 for different G4s remain unknown. Here, using CD spectroscopy and 83 G4 motifs from S. cerevisiae ranging in length from 30 to 60 nucleotides, we first show that G4 structures can be formed with a broad range of loop sizes in vitro and that a parallel conformation is favored. Using single-molecule FRET analysis, we then systematically addressed Pif1-mediated unwinding of various G4s and found that Pif1 is sensitive to G4 stability. Moreover, Pif1 preferentially unfolded antiparallel G4s rather than parallel G4s having similar stability. Furthermore, our results indicate that most G4 structures in S. cerevisiae sequences have long loops and can be efficiently unfolded by Pif1 because of their low stability. However, we also found that G4 structures with short loops can be barely unfolded. This study highlights the formidable capability of Pif1 to resolve the majority of G4s in S. cerevisiae sequences, narrows the fractions of G4s that may be challenging for genomic stability, and provides a framework for understanding the influence of different G4s on genomic stability via their processing by Pif1.

Section: Pif1 Unfolds Different G4 Structures With Different Efficiensupporting

confidence: 89%

Section: Modulation Of Pif1 Unwinding Activity By G-quadruplex Dnamentioning

confidence: 99%

DNA-unwinding activity of Saccharomyces cerevisiae Pif1 is modulated by thermal stability, folding conformation, and loop lengths of G-quadruplex DNA

Qingman

Journal of Biological Chemistry

et al. 2018

Self Cite

“…While the enzymatic functions of the Pif1 helicase have been extensively characterized, the precise mechanisms by which the activities of this helicase are coordinated to impact a variety of nuclear DNA transactions remain unknown [48][49][50][51][52]. Pif1's cellular abundance is predicted to be low [53], and aberrant Pif1 levels in the cell lead to deleterious effects.…”

Section: Discussionmentioning

confidence: 99%

Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

Ononye

Sausen

Balakrishnan

et al. 2020

Preprint

ABSTRACTIn S. cerevisiae, the Pif1 helicase functions to impact both nuclear and mitochondrial DNA replication and repair processes. Pif1 is a 5’-3’ helicase, which preferentially unwinds RNA-DNA hybrids and resolves G-quadruplex structures. Further, regulation of Pif1 by phosphorylation negatively impacts its interaction with telomerase during double strand break repair. Here, we report that in addition to phosphorylation, Pif1 is also modified by lysine acetylation, which influences both its cellular and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 (Esa1) and deacetylase Rpd3 are primarily responsible for dynamically acetylating nuclear Pif1. Mass spectrometry analysis revealed that Pif1 was modified throughout the protein’s sequence on the N-terminus (K118, K129), helicase domain (K525, K639, K725), and C-terminus (K800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N-terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase activity, while maintaining its substrate preferences. Additionally, both the ATPase and DNA binding activities of Pif1 were stimulated upon acetylation. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose an acetylation-based model for the regulation of Pif1 activities, addressing how this post translational modification can influence its role as a key player in a multitude of DNA transactions vital to the maintenance of genome integrity.

“…Although the focus of structural studies has historically been to elucidate the molecular mechanism for unwinding dsDNA, in 2018 two papers appeared which addressed the structural elements and molecular details for resolving G-quadruplex DNA by the S. cerevisiae Pif1 [201] and C. sakazakii RecQ [202] helicases. This area of research is likely to blossom with the growing interest in the roles of unconventionally structured DNA in genome biology.…”

Section: Dna Helicase Protein Structuresmentioning

confidence: 99%

History of DNA Helicases

Brosh

Matson

2020

Genes

Since the discovery of the DNA double helix, there has been a fascination in understanding the molecular mechanisms and cellular processes that account for: (i) the transmission of genetic information from one generation to the next and (ii) the remarkable stability of the genome. Nucleic acid biologists have endeavored to unravel the mysteries of DNA not only to understand the processes of DNA replication, repair, recombination, and transcription but to also characterize the underlying basis of genetic diseases characterized by chromosomal instability. Perhaps unexpectedly at first, DNA helicases have arisen as a key class of enzymes to study in this latter capacity. From the first discovery of ATP-dependent DNA unwinding enzymes in the mid 1970’s to the burgeoning of helicase-dependent pathways found to be prevalent in all kingdoms of life, the story of scientific discovery in helicase research is rich and informative. Over four decades after their discovery, we take this opportunity to provide a history of DNA helicases. No doubt, many chapters are left to be written. Nonetheless, at this juncture we are privileged to share our perspective on the DNA helicase field – where it has been, its current state, and where it is headed.