Structural mechanism underpinning Thermus oshimai Pif1-mediated G-quadruplex unfolding.

G-quadruplexes (G4s) are unusual stable DNA structures that cause genomic instability. To overcome the potential barriers formed by G4s, cells have evolved different families of proteins that unfold G4s. Pif1 is a DNA helicase from superfamily 1 (SF1) conserved from bacteria to humans with high G4-unwinding activity. Here, we present the first X-ray crystal structure of the Thermus oshimai Pif1 (ToPif1) complexed with a G4. Our structure reveals that ToPif1 recognizes the entire native G4 via a cluster of amino acids at domains 1B/2B which constitute a G4-Recognizing Surface (GRS). The overall structure of the G4 maintains its three-layered propeller-type G4 topology, without significant reorganization of G-tetrads upon protein binding. The three G-tetrads in G4 are recognized by GRS residues mainly through electrostatic, ionic interactions, and hydrogen bonds formed between the GRS residues and the ribose-phosphate backbone. Compared with previously solved structures of SF2 helicases in complex with G4, our structure reveals how helicases from distinct superfamilies adopt different strategies for recognizing and unfolding G4s.

Structural and functional studies of SF1B Pif1 from Thermus oshimai reveal dimerization-induced helicase inhibition.

Pif1 is an SF1B helicase that is evolutionarily conserved from bacteria to humans and plays multiple roles in maintaining genome stability in both nucleus and mitochondria. Though highly conserved, Pif1 family harbors a large mechanistic diversity. Here, we report crystal structures of Thermus oshimai Pif1 (ToPif1) alone and complexed with partial duplex or single-stranded DNA. In the apo state and in complex with a partial duplex DNA, ToPif1 is monomeric with its domain 2B/loop3 adopting a closed and an open conformation, respectively. When complexed with a single-stranded DNA, ToPif1 forms a stable dimer with domain 2B/loop3 shifting to a more open conformation. Single-molecule and biochemical assays show that domain 2B/loop3 switches repetitively between the closed and open conformations when a ToPif1 monomer unwinds DNA and, in contrast with other typical dimeric SF1A helicases, dimerization has an inhibitory effect on its helicase activity. This mechanism is not general for all Pif1 helicases but illustrates the diversity of regulation mechanisms among different helicases. It also raises the possibility that although dimerization results in activation for SF1A helicases, it may lead to inhibition for some of the other uncharacterized SF1B helicases, an interesting subject warranting further studies.

Quantitative and real-time measurement of helicase-mediated intra-stranded G4 unfolding in bulk fluorescence stopped-flow assays.

G-Quadruplexes (G4s) are thermodynamically stable, compact, and poorly hydrated structures that pose a potent obstacle for chromosome replication and gene expression, and requiring resolution by helicases in a cell. Bulk stopped-flow fluorescence assays have provided many mechanistic insights into helicase-mediated duplex DNA unwinding. However, to date, detailed studies on intramolecular G-quadruplexes similar or comparable with those used for studying duplex DNA are still lacking. Here, we describe a method for the direct and quantitative measurement of helicase-mediated intramolecular G-quadruplex unfolding in real time. We designed a series of site-specific fluorescently double-labeled intramolecular G4s and screened appropriate substrates to characterize the helicase-mediated G4 unfolding. With the developed method, we determined, for the first time to our best knowledge, the unfolding and refolding constant of G4 (≈ 5 s-1), and other relative parameters under single-turnover experimental conditions in the presence of G4 traps. Our approach not only provides a new paradigm for characterizing helicase-mediated intramolecular G4 unfolding using stopped-flow assays but also offers a way to screen for inhibitors of G4 unfolding helicases as therapeutic drug targets. Graphical abstract.

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

The Saccharomyces cerevisiae Pif1 protein (ScPif1p) is the prototypical member of the Pif1 family of DNA helicases. ScPif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and suppresses genome instability at G-quadruplex motifs. Here, we report the crystal structures of a truncated ScPif1p (ScPif1p237-780) in complex with different ssDNAs. Our results have revealed that a yeast-specific insertion domain protruding from the 2B domain folds as a bundle bearing an α-helix, α16. The α16 helix regulates the helicase activities of ScPif1p through interactions with the previously identified loop3. Furthermore, a biologically relevant dimeric structure has been identified, which can be further specifically stabilized by G-quadruplex DNA. Basing on structural analyses and mutational studies with DNA binding and unwinding assays, a potential G-quadruplex DNA binding site in ScPif1p monomers is suggested. Our results also show that ScPif1p uses the Q-motif to preferentially hydrolyze ATP, and a G-rich tract is preferentially recognized by more residues, consistent with previous biochemical observations. These findings provide a structural and mechanistic basis for understanding the multifunctional ScPif1p.

Crystal structure of Escherichia coli DEAH/RHA helicase HrpB.

RNA helicases are almost ubiquitous important enzymes that take part in multiple aspects of RNA metabolism. Prokaryotes encode fewer RNA helicases than eukaryotes, suggesting that individual prokaryotic RNA helicases may take on multiple roles. The specific functions and molecular mechanisms of bacterial DEAH/RHA helicases are poorly understood, and no structures are available of these bacterial enzymes. Here, we report the first crystal structure of the DEAH/RHA helicase HrpB of Escherichia coli in a complex with ADP•AlF4. It showed an atypical globular structure, consisting of two RecA domains, an HA2 domain and an OB domain, similar to eukaryotic DEAH/RHA helicases. Notably, it showed a unique C-terminal extension that has never been reported before. Activity assays indicated that EcHrpB binds RNA but not DNA, and does not exhibit unwinding activity in vitro. Thus, within cells, the EcHrpB may function in helicase activity-independent RNA metabolic processes.

Crystal structures of the BsPif1 helicase reveal that a major movement of the 2B SH3 domain is required for DNA unwinding.

Pif1 helicases are ubiquitous members of the SF1B family and are essential for maintaining genome stability. It was speculated that Pif1-specific motifs may fold in specific structures, conferring distinct activities upon it. Here, we report the crystal structures of the Pif1 helicase from Bacteroides spp with and without adenosine triphosphate (ATP) analog/ssDNA. BsPif1 shares structural similarities with RecD2 and Dda helicases but has specific features in the 1B and 2B domains. The highly conserved Pif1 family specific sequence motif interacts with and constraints a putative pin-loop in domain 1B in a precise conformation. More importantly, we found that the 2B domain which contains a specific extended hairpin undergoes a significant rotation and/or movement upon ATP and DNA binding, which is absolutely required for DNA unwinding. We therefore propose a mechanism for DNA unwinding in which the 2B domain plays a predominant role. The fact that the conformational change regulates Pif1 activity may provide insight into the puzzling observation that Pif1 becomes highly processive during break-induced replication in association with Polδ, while the isolated Pif1 has low processivity.

Stimulation of ATP Hydrolysis by ssDNA Provides the Necessary Mechanochemical Energy for G4 Unfolding.

The G-quadruplex (G4) is a distinct geometric and electrophysical structure compared to classical double-stranded DNA, and its stability can impede essential cellular processes such as replication, transcription, and translation. This study focuses on the BsPif1 helicase, revealing its ability to bind independently to both single-stranded DNA (ssDNA) and G4 structures. The unfolding activity of BsPif1 on G4 relies on the presence of a single tail chain, and the covalent continuity between the single tail chain and the G4's main chain is necessary for efficient G4 unwinding. This suggests that ATP hydrolysis-driven ssDNA translocation exerts a pull force on G4 unwinding. Molecular dynamics simulations identified a specific region within BsPif1 that contains five crucial amino acid sites responsible for G4 binding and unwinding. A "molecular wire stripper" model is proposed to explain BsPif1's mechanism of G4 unwinding. These findings provide a new theoretical foundation for further exploration of the G4 development mechanism in Pif1 family helicases.

Endogenous Bos taurus RECQL is predominantly monomeric and more active than oligomers.

There is broad consensus that RecQ family helicase is a high-order oligomer that dissociates into a dimer upon ATP binding. This conclusion is based mainly on studies of highly purified recombinant proteins, and the oligomeric states of RecQ helicases in living cells remain unknown. We show here that, in contrast to current models, monomeric RECQL helicase is more abundant than oligomer/dimer forms in living cells. Further characterization of endogenous BtRECQL and isolated monomeric BtRECQL using various approaches demonstrates that both endogenous and recombinant monomeric BtRECQL effectively function as monomers, displaying higher helicase and ATPase activities than dimers and oligomers. Furthermore, monomeric BtRECQL unfolds intramolecular G-quadruplex DNA as efficiently as human RECQL and BLM helicases. These discoveries have implications for understanding endogenous RECQL oligomeric structures and their regulation. It is worth revisiting oligomeric states of the other members of the RecQ family helicases in living cells.

Molecular Mechanistic Insights into Drosophila DHX36-Mediated G-Quadruplex Unfolding: A Structure-Based Model.

Helicase DHX36 plays essential roles in cell development and differentiation at least partially by resolving G-quadruplex (G4) structures. Here we report crystal structures of the Drosophila homolog of DHX36 (DmDHX36) in complex with RNA and a series of DNAs. By combining structural, small-angle X-ray scattering, molecular dynamics simulation, and single-molecule fluorescence studies, we revealed that positively charged amino acids in RecA2 and OB-like domains constitute an elaborate structural pocket at the nucleic acid entrance, in which negatively charged G4 DNA is tightly bound and partially destabilized. The G4 DNA is then completely unfolded through the 3'-5' translocation activity of the helicase. Furthermore, crystal structures and DNA binding assays show that G-rich DNA is preferentially recognized and in the presence of ATP, specifically bound by DmDHX36, which may cooperatively enhance the G-rich DNA translocation and G4 unfolding. On the basis of these results, a conceptual G4 DNA-resolving mechanism is proposed.