Two residues in the DNA binding site of Pif1 helicase are essential for nuclear functions but dispensable for mitochondrial respiratory growth.

Pif1 helicase functions in both the nucleus and mitochondria. Pif1 tightly couples ATP hydrolysis, single-stranded DNA translocation, and duplex DNA unwinding. We investigated two Pif1 variants (F723A and T464A) that have each lost one site of interaction of the protein with the DNA substrate. Both variants exhibit minor reductions in affinity for DNA and ATP hydrolysis but have impaired DNA unwinding activity. However, these variants translocate on single-stranded DNA faster than the wildtype enzyme and can slide on the DNA substrate in an ATP-independent manner. This suggests they have lost their grip on the DNA, interfering with coupling ATP hydrolysis to translocation and unwinding. Yeast expressing these variants have increased gross chromosomal rearrangements, increased telomere length, and can overcome the lethality of dna2Δ, similar to phenotypes of yeast lacking Pif1. However, unlike pif1Δ mutants, they are viable on glycerol containing media and maintain similar mitochondrial DNA copy numbers as Pif1 wildtype. Overall, our data indicate that a tight grip of the trailing edge of the Pif1 enzyme on the DNA couples ATP hydrolysis to DNA translocation and DNA unwinding. This tight grip appears to be essential for the Pif1 nuclear functions tested but is dispensable for mitochondrial respiratory growth.

Excessive excision of correct nucleotides during DNA synthesis explained by replication hurdles.

The proofreading exonuclease activity of replicative DNA polymerase excises misincorporated nucleotides during DNA synthesis, but these events are rare. Therefore, we were surprised to find that T7 replisome excised nearly 7% of correctly incorporated nucleotides during leading and lagging strand syntheses. Similar observations with two other DNA polymerases establish its generality. We show that excessive excision of correctly incorporated nucleotides is not due to events such as processive degradation of nascent DNA or spontaneous partitioning of primer-end to the exonuclease site as a "cost of proofreading". Instead, we show that replication hurdles, including secondary structures in template, slowed helicase, or uncoupled helicase-polymerase, increase DNA reannealing and polymerase backtracking, and generate frayed primer-ends that are shuttled to the exonuclease site and excised efficiently. Our studies indicate that active-site shuttling occurs at a high frequency, and we propose that it serves as a proofreading mechanism to protect primer-ends from mutagenic extensions.

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.

Hepatitis C virus nonstructural protein NS3 unfolds viral G-quadruplex RNA structures.

Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the nonstructural proteins of HCV, NS3 (nonstructural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)-forming sequences in the HCV-positive and HCV-negative strands. We performed fluorescence anisotropy binding, G4 reporter duplex unwinding, and G4RNA trapping assays to determine the binding and G4 unfolding activity of NS3. Our data suggest that NS3 can unfold the conserved G4 structures present within the genome and the negative strand of HCV. Additionally, we found the activity of NS3 on a G4RNA was reduced significantly in the presence of a G4 ligand. The ability of NS3 to unfold HCV G4RNA could imply a novel biological role of the viral helicase in replication.

Primary acute lymphoblastic leukemia cells are susceptible to microtubule depolymerization in G1 and M phases through distinct cell death pathways.

Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs.

G-Quadruplex loops regulate PARP-1 enzymatic activation.

G-Quadruplexes are non-B form DNA structures present at regulatory regions in the genome, such as promoters of proto-oncogenes and telomeres. The prominence in such sites suggests G-quadruplexes serve an important regulatory role in the cell. Indeed, oxidized G-quadruplexes found at regulatory sites are regarded as epigenetic elements and are associated with an interlinking of DNA repair and transcription. PARP-1 binds damaged DNA and non-B form DNA, where it covalently modifies repair enzymes or chromatin-associated proteins respectively with poly(ADP-ribose) (PAR). PAR serves as a signal in regulation of transcription, chromatin remodeling, and DNA repair. PARP-1 is known to bind G-quadruplexes with stimulation of enzymatic activity. We show that PARP-1 binds several G-quadruplex structures with nanomolar affinities, but only a subset promote PARP-1 activity. The G-quadruplex forming sequence found in the proto-oncogene c-KIT promoter stimulates enzymatic activity of PARP-1. The loop-forming characteristics of the c-KIT G-quadruplex sequence regulate PARP-1 catalytic activity, whereas eliminating these loop features reduces PARP-1 activity. Oxidized G-quadruplexes that have been suggested to form unique, looped structures stimulate PARP-1 activity. Our results support a functional interaction between PARP-1 and G-quadruplexes. PARP-1 enzymatic activation by G-quadruplexes is dependent on the loop features and the presence of oxidative damage.

Mitochondrial genetic variation is enriched in G-quadruplex regions that stall DNA synthesis in vitro.

As the powerhouses of the eukaryotic cell, mitochondria must maintain their genomes which encode proteins essential for energy production. Mitochondria are characterized by guanine-rich DNA sequences that spontaneously form unusual three-dimensional structures known as G-quadruplexes (G4). G4 structures can be problematic for the essential processes of DNA replication and transcription because they deter normal progression of the enzymatic-driven processes. In this study, we addressed the hypothesis that mitochondrial G4 is a source of mutagenesis leading to base-pair substitutions. Our computational analysis of 2757 individual genomes from two Italian population cohorts (SardiNIA and InCHIANTI) revealed a statistically significant enrichment of mitochondrial mutations within sequences corresponding to stable G4 DNA structures. Guided by the computational analysis results, we designed biochemical reconstitution experiments and demonstrated that DNA synthesis by two known mitochondrial DNA polymerases (Pol γ, PrimPol) in vitro was strongly blocked by representative stable G4 mitochondrial DNA structures, which could be overcome in a specific manner by the ATP-dependent G4-resolving helicase Pif1. However, error-prone DNA synthesis by PrimPol using the G4 template sequence persisted even in the presence of Pif1. Altogether, our results suggest that genetic variation is enriched in G-quadruplex regions that impede mitochondrial DNA replication.

Direct quantification of the translocation activities of Saccharomyces cerevisiae Pif1 helicase.

Saccharomyces cerevisiae Pif1 (ScPif1) is known as an ATP-dependent DNA helicase that plays critical roles in a number of important biological processes such as DNA replication, telomere maintenance and genome stability maintenance. Besides its DNA helicase activity, ScPif1 is also known as a single-stranded DNA (ssDNA) translocase, while how ScPif1 translocates on ssDNA is unclear. Here, by measuring the translocation activity of individual ScPif1 molecules on ssDNA extended by mechanical force, we identified two distinct types of ssDNA translocation. In one type, ScPif1 moves along the ssDNA track with a rate of ∼140 nt/s in 100 µM ATP, whereas in the other type, ScPif1 is immobilized to a fixed location of ssDNA and generates ssDNA loops against force. Between the two, the mobile translocation is the major form at nanomolar ScPif1 concentrations although patrolling becomes more frequent at micromolar concentrations. Together, our results suggest that ScPif1 translocates on extended ssDNA in two distinct modes, primarily in a 'mobile' manner.

Pif1 helicase unfolding of G-quadruplex DNA is highly dependent on sequence and reaction conditions.

In addition to unwinding double-stranded nucleic acids, helicase activity can also unfold noncanonical structures such as G-quadruplexes. We previously characterized Pif1 helicase catalyzed unfolding of parallel G-quadruplex DNA. Here we characterized unfolding of the telomeric G-quadruplex, which can fold into antiparallel and mixed hybrid structures and found significant differences. Telomeric DNA sequences are unfolded more readily than the parallel quadruplex formed by the c-MYC promoter in K+ Furthermore, we found that under conditions in which the telomeric quadruplex is less stable, such as in Na+, Pif1 traps thermally melted quadruplexes in the absence of ATP, leading to the appearance of increased product formation under conditions in which the enzyme is preincubated with the substrate. Stable telomeric G-quadruplex structures were unfolded in a stepwise manner at a rate slower than that of duplex DNA unwinding; however, the slower dissociation from G-quadruplexes compared with duplexes allowed the helicase to traverse more nucleotides than on duplexes. Consistent with this, the rate of ATP hydrolysis on the telomeric quadruplex DNA was reduced relative to that on single-stranded DNA (ssDNA), but less quadruplex DNA was needed to saturate ATPase activity. Under single-cycle conditions, telomeric quadruplex was unfolded by Pif1, but for the c-MYC quadruplex, unfolding required multiple helicase molecules loaded onto the adjacent ssDNA. Our findings illustrate that Pif1-catalyzed unfolding of G-quadruplex DNA is highly dependent on the specific sequence and the conditions of the reaction, including both the monovalent cation and the order of addition.

N-Naphthoyl-substituted indole thio-barbituric acid analogs inhibit the helicase activity of the hepatitis C virus NS3.

The hepatitis C virus (HCV) represents a substantial threat to human health worldwide. The virus expresses a dual-function protein, NS3 having both protease and RNA helicase activities that are essential for productive viral replication and sustained infections. While viral protease and polymerase inhibitors have shown great successes in treating chronic HCV infections, drugs that specifically target the helicase activity have not advanced. A robust and quantitative 96-well plate-based fluorescent DNA unwinding assay was used to screen a class of indole thio-barbituric acid (ITBA) analogs using the full-length, recombinant HCV NS3, and identified three naphthoyl-containing analogs that efficiently inhibited NS3 helicase activity in a dose-dependent manner, with observed IC50 values of 21-24 µM. Standard gel electrophoresis helicase assays using radiolabeled duplex DNA and RNA NS3 substrates confirmed the inhibition of NS3 unwinding activity. Subsequent anisotropy measurements demonstrated that the candidate compounds did not disrupt NS3 binding to nucleic acids. Additionally, the rate of ATP hydrolysis and the protease activity were also not affected by the inhibitors. Thus, these results indicate that the three ITBA analogs containing N-naphthoyl moieties are the foundation of a potential series of small molecules capable of inhibiting NS3 activity via a novel interaction with the helicase domain that prevents the productive unwinding of nucleic acid substrates, and may represent the basis for a new class of therapeutic agents with the potential to aid in the treatment and eradication of hepatitis C virus.

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 catch and release program for single-stranded DNA.

Uncovering the mechanisms by which single-stranded binding proteins both protect and expose single-stranded DNA has important implications for our understanding of DNA replication and repair. A new study serves up a master class in developing a full kinetic model for one such protein, mtSSB, showing how DNA can be reeled in and set free to control accessibility.

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.

Structure and function of Pif1 helicase.

Pif1 family helicases have multiple roles in the maintenance of nuclear and mitochondrial DNA in eukaryotes. Saccharomyces cerevisiae Pif1 is involved in replication through barriers to replication, such as G-quadruplexes and protein blocks, and reduces genetic instability at these sites. Another Pif1 family helicase in S. cerevisiae, Rrm3, assists in fork progression through replication fork barriers at the rDNA locus and tRNA genes. ScPif1 (Saccharomyces cerevisiae Pif1) also negatively regulates telomerase, facilitates Okazaki fragment processing, and acts with polymerase δ in break-induced repair. Recent crystal structures of bacterial Pif1 helicases and the helicase domain of human PIF1 combined with several biochemical and biological studies on the activities of Pif1 helicases have increased our understanding of the function of these proteins. This review article focuses on these structures and the mechanism(s) proposed for Pif1's various activities on DNA.

Protein-protein interaction analysis for functional characterization of helicases.

Helicases are enzymes involved in nucleic acid metabolism, playing major roles in replication, transcription, and repair. Defining helicases oligomerization state and transient and persistent protein interactions is essential for understanding of their function. In this article we review current methods for the protein-protein interaction analysis, and discuss examples of its application to the study of helicases: Pif1 and DDX3. Proteomics methods are our main focus - affinity pull-downs and chemical cross-linking followed by mass spectrometry. We review advantages and limitations of these methods and provide general guidelines for their implementation in the functional analysis of helicases.

A parallel quadruplex DNA is bound tightly but unfolded slowly by pif1 helicase.

DNA sequences that can form intramolecular quadruplex structures are found in promoters of proto-oncogenes. Many of these sequences readily fold into parallel quadruplexes. Here we characterize the ability of yeast Pif1 to bind and unfold a parallel quadruplex DNA substrate. We found that Pif1 binds more tightly to the parallel quadruplex DNA than single-stranded DNA or tailed duplexes. However, Pif1 unwinding of duplexes occurs at a much faster rate than unfolding of a parallel intramolecular quadruplex. Pif1 readily unfolds a parallel quadruplex DNA substrate in a multiturnover reaction and also generates some product under single cycle conditions. The rate of ATP hydrolysis by Pif1 is reduced when bound to a parallel quadruplex compared with single-stranded DNA. ATP hydrolysis occurs at a faster rate than quadruplex unfolding, indicating that some ATP hydrolysis events are non-productive during unfolding of intramolecular parallel quadruplex DNA. However, product eventually accumulates at a slow rate.

Simultaneous binding to the tracking strand, displaced strand and the duplex of a DNA fork enhances unwinding by Dda helicase.

Interactions between helicases and the tracking strand of a DNA substrate are well-characterized; however, the role of the displaced strand is a less understood characteristic of DNA unwinding. Dda helicase exhibited greater processivity when unwinding a DNA fork compared to a ss/ds DNA junction substrate. The lag phase in the unwinding progress curve was reduced for the forked DNA compared to the ss/ds junction. Fewer kinetic steps were required to unwind the fork compared to the ss/ds junction, suggesting that binding to the fork leads to disruption of the duplex. DNA footprinting confirmed that interaction of Dda with a fork leads to two base pairs being disrupted whereas no disruption of base pairing was observed with the ss/ds junction. Neutralization of the phosphodiester backbone resulted in a DNA-footprinting pattern similar to that observed with the ss/ds junction, consistent with disruption of the interaction between Dda and the displaced strand. Several basic residues in the 1A domain which were previously proposed to bind to the incoming duplex DNA were replaced with alanines, resulting in apparent loss of interaction with the duplex. Taken together, these results suggest that Dda interaction with the tracking strand, displaced strand and duplex coordinates DNA unwinding.

Melting of Duplex DNA in the Absence of ATP by the NS3 Helicase Domain through Specific Interaction with a Single-Strand/Double-Strand Junction.

Helicases unwind double-stranded nucleic acids, remove secondary structures from single-stranded nucleic acids, and remove proteins bound to nucleic acids. For many helicases, the mechanisms for these different functions share the ability to translocate with a directional bias as a result of ATP binding and hydrolysis. Nonstructural protein 3 (NS3) is an essential enzyme expressed by the hepatitis C virus (HCV) and is known to catalyze the unwinding of both DNA and RNA substrates in a 3'-to-5' direction. We investigated the role of nucleic acid binding in the unwinding mechanism by examining ATP-independent unwinding. We observed that even in the absence of ATP, the NS3 helicase domain (NS3h) unwound duplexes only when they contained a 3'-tail (i.e., 3'-to-5' directionality). Blunt-ended duplexes and 5'-tailed duplexes were not melted even in the presence of a large excess concentration of the protein. NS3h was found to diffuse rapidly along single-stranded DNA at a rate of 30 nucleotides(2) s(-1). Upon encountering an appropriate single-strand/double-strand (ss/ds) junction, NS3h slowly melted the duplex under conditions with an excess protein concentration relative to DNA concentration. When a biotin-streptavidin block was placed into the ssDNA region, no melting of DNA was observed, suggesting that NS3h must diffuse along the ssDNA, and that the streptavidin blocked the diffusion. We conclude that the specific interaction between NS3h and the ss/dsDNA junction, coupled with diffusion, allows binding energy to melt duplex DNA with a directional bias. Alternatively, we found that the full-length NS3 protein did not exhibit strict directionality and was dependent on duplex DNA length. NS3 was able to unwind the duplex even in the presence of the biotin-streptavidin block. We propose a noncanonical model of unwinding for NS3 in which the enzyme binds directly to the duplex via protein-protein interactions to melt the substrate.