Pif1 Helicase Mediates Remodeling of Protein-Nucleic Acid Complexes by Promoting Dissociation of Sub1 from G-Quadruplex DNA and Cdc13 from G-Rich Single-Stranded DNA.

Pif1 is a molecular motor enzyme that is conserved from yeast to mammals. It translocates on ssDNA with a directional bias (5' → 3') and unwinds duplexes using the energy obtained from ATP hydrolysis. Pif1 is involved in dsDNA break repair, resolution of G-quadruplex (G4) structures, negative regulation of telomeres, and Okazaki fragment maturation. An important property of this helicase is to exert force and disrupt protein-DNA complexes, which may otherwise serve as barriers to various cellular pathways. Previously, Pif1 was reported to displace streptavidin from biotinylated DNA, Rap1 from telomeric DNA, and telomerase from DNA ends. Here, we have investigated the ability of S. cerevisiae Pif1 helicase to disrupt protein barriers from G4 and telomeric sites. Yeast chromatin-associated transcription coactivator Sub1 was characterized as a G4 binding protein. We found evidence for a physical interaction between Pif1 helicase and Sub1 protein. Here, we demonstrate that Pif1 is capable of catalyzing the disruption of Sub1-bound G4 structures in an ATP-dependent manner. We also investigated Pif1-mediated removal of yeast telomere-capping protein Cdc13 from DNA ends. Cdc13 exhibits a high-affinity interaction with an 11-mer derived from the yeast telomere sequence. Our results show that Pif1 uses its translocase activity to enhance the dissociation of this telomere-specific protein from its binding site. The rate of dissociation increased with an increase in the helicase loading site length. Additionally, we examined the biochemical mechanism for Pif1-catalyzed protein displacement by mutating the sequence of the telomeric 11-mer on the 5'-end and the 3'-end. The results support a model whereby Pif1 disrupts Cdc13 from the ssDNA in steps.

Yeast Sub1 and human PC4 are G-quadruplex binding proteins that suppress genome instability at co-transcriptionally formed G4 DNA.

G-quadruplex or G4 DNA is a non-B secondary DNA structure consisting of a stacked array of guanine-quartets that can disrupt critical cellular functions such as replication and transcription. When sequences that can adopt Non-B structures including G4 DNA are located within actively transcribed genes, the reshaping of DNA topology necessary for transcription process stimulates secondary structure-formation thereby amplifying the potential for genome instability. Using a reporter assay designed to study G4-induced recombination in the context of an actively transcribed locus in Saccharomyces cerevisiae, we tested whether co-transcriptional activator Sub1, recently identified as a G4-binding factor, contributes to genome maintenance at G4-forming sequences. Our data indicate that, upon Sub1-disruption, genome instability linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly augmented and that its highly conserved DNA binding domain or the human homolog PC4 is sufficient to suppress G4-associated genome instability. We also show that Sub1 interacts specifically with co-transcriptionally formed G4 DNA in vivo and that yeast cells become highly sensitivity to G4-stabilizing chemical ligands by the loss of Sub1. Finally, we demonstrate the physical and genetic interaction of Sub1 with the G4-resolving helicase Pif1, suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA.

A biochemical and biophysical model of G-quadruplex DNA recognition by positive coactivator of transcription 4.

DNA sequences that are guanine-rich have received considerable attention because of their potential to fold into a secondary, four-stranded DNA structure termed G-quadruplex (G4), which has been implicated in genomic instability and some human diseases. We have previously identified positive coactivator of transcription (PC4), a single-stranded DNA (ssDNA)-binding protein, as a novel G4 interactor. Here, to expand on these previous observations, we biochemically and biophysically characterized the interaction between PC4 and G4DNA. PC4 can bind alternative G4DNA topologies with a low nanomolar Kd value of ∼2 nm, similar to that observed for ssDNA. In consideration of the different structural features between G4DNA and ssDNA, these binding data indicated that PC4 can interact with G4DNA in a manner distinct from ssDNA. The stoichiometry of the PC4-G4 complex was 1:1 for PC4 dimer:G4 substrate. PC4 did not enhance the rate of folding of G4DNA, and formation of the PC4-G4DNA complex did not result in unfolding of the G4DNA structure. We assembled a G4DNA structure flanked by duplex DNA. We find that PC4 can interact with this G4DNA, as well as the complementary C-rich strand. Molecular docking simulations and DNA footprinting experiments suggest a model where a PC4 dimer accommodates the DNA with one monomer on the G4 strand and the second monomer bound to the C-rich strand. Collectively, these data provide a novel mode of PC4 binding to a DNA secondary structure that remains within the framework of the model for binding to ssDNA. Additionally, consideration of the PC4-G4DNA interaction could provide insight into the biological functions of PC4, which remain incompletely understood.

Yeast Helicase Pif1 Unwinds RNA:DNA Hybrids with Higher Processivity than DNA:DNA Duplexes.

Saccharomyces cerevisiae Pif1, an SF1B helicase, has been implicated in both mitochondrial and nuclear functions. Here we have characterized the preference of Pif1 for RNA:DNA heteroduplexes in vitro by investigating several kinetic parameters associated with unwinding. We show that the preferential unwinding of RNA:DNA hybrids is due to neither specific binding nor differences in the rate of strand separation. Instead, Pif1 is capable of unwinding RNA:DNA heteroduplexes with moderately greater processivity compared with its duplex DNA:DNA counterparts. This higher processivity of Pif1 is attributed to slower dissociation from RNA:DNA hybrids. Biologically, this preferential role of the helicase may contribute to its functions at both telomeric and nontelomeric sites.

Evidence That G-quadruplex DNA Accumulates in the Cytoplasm and Participates in Stress Granule Assembly in Response to Oxidative Stress.

Cells engage numerous signaling pathways in response to oxidative stress that together repair macromolecular damage or direct the cell toward apoptosis. As a result of DNA damage, mitochondrial DNA or nuclear DNA has been shown to enter the cytoplasm where it binds to "DNA sensors," which in turn initiate signaling cascades. Here we report data that support a novel signaling pathway in response to oxidative stress mediated by specific guanine-rich sequences that can fold into G-quadruplex DNA (G4DNA). In response to oxidative stress, we demonstrate that sequences capable of forming G4DNA appear at increasing levels in the cytoplasm and participate in assembly of stress granules. Identified proteins that bind to endogenous G4DNA in the cytoplasm are known to modulate mRNA translation and participate in stress granule formation. Consistent with these findings, stress granule formation is known to regulate mRNA translation during oxidative stress. We propose a signaling pathway whereby cells can rapidly respond to DNA damage caused by oxidative stress. Guanine-rich sequences that are excised from damaged genomic DNA are proposed to enter the cytoplasm where they can regulate translation through stress granule formation. This newly proposed role for G4DNA provides an additional molecular explanation for why such sequences are prevalent in the human genome.

Yeast Pif1 helicase exhibits a one-base-pair stepping mechanism for unwinding duplex DNA.

Kinetic analysis of the DNA unwinding and translocation activities of helicases is necessary for characterization of the biochemical mechanism(s) for this class of enzymes. Saccharomyces cerevisiae Pif1 helicase was characterized using presteady state kinetics to determine rates of DNA unwinding, displacement of streptavidin from biotinylated DNA, translocation on single-stranded DNA (ssDNA), and ATP hydrolysis activities. Unwinding of substrates containing varying duplex lengths was fit globally to a model for stepwise unwinding and resulted in an unwinding rate of ∼75 bp/s and a kinetic step size of 1 base pair. Pif1 is capable of displacing streptavidin from biotinylated oligonucleotides with a linear increase in the rates as the length of the oligonucleotides increased. The rate of translocation on ssDNA was determined by measuring dissociation from varying lengths of ssDNA and is essentially the same as the rate of unwinding of dsDNA, making Pif1 an active helicase. The ATPase activity of Pif1 on ssDNA was determined using fluorescently labeled phosphate-binding protein to measure the rate of phosphate release. The quantity of phosphate released corresponds to a chemical efficiency of 0.84 ATP/nucleotides translocated. Hence, when all of the kinetic data are considered, Pif1 appears to move along DNA in single nucleotide or base pair steps, powered by hydrolysis of 1 molecule of ATP.

DEAD-box RNA helicases Dbp2, Ded1 and Mss116 bind to G-quadruplex nucleic acids and destabilize G-quadruplex RNA.

We identified 29 G-quadruplex binding proteins by affinity purification and quantitative LC-MS/MS. We demonstrated that the DEAD-box RNA helicases Dbp2, Ded1 and Mss116 preferentially bind to G-quadruplex nucleic acids in vitro and destabilize RNA quadruplexes, suggesting new potential roles for these helicases in disruption of quadruplex structures in RNA.

Yeast transcription co-activator Sub1 and its human homolog PC4 preferentially bind to G-quadruplex DNA.

Using a G-quadruplex bait, we identified the transcription co-activator Sub1 as a G-quadruplex binding protein by quantitative LC-MS/MS and demonstrated in vivo G-quadruplex binding by ChIP. In vitro, Sub1, and its human homolog PC4, bind preferentially to G-quadruplexes. This provides a possible mechanism by which G-quadruplexes can influence gene transcription.