The catalytic triad of rice NARROW LEAF1 involves H234.

NARROW LEAF1 (NAL1) exerts a multifaceted influence on leaf morphology and crop yield. Recent crystal study proposed that histidine 233 (H233) is part of the catalytic triad. Here we report that unlike suggested previously, H234 instead of H233 is a component of the catalytic triad alongside residues D291 and S385 in NAL1. Remarkably, residue 233 unexpectedly plays a pivotal role in regulating NAL1's proteolytic activity. These findings establish a strong foundation for utilizing NAL1 in breeding programs aimed at improving crop yield.

Structural insights into the N-terminal APHB domain of HrpA: mediating canonical and i-motif recognition.

RNA helicases function as versatile enzymes primarily responsible for remodeling RNA secondary structures and organizing ribonucleoprotein complexes. In our study, we conducted a systematic analysis of the helicase-related activities of Escherichia coli HrpA and presented the structures of both its apo form and its complex bound with both conventional and non-canonical DNAs. Our findings reveal that HrpA exhibits NTP hydrolysis activity and binds to ssDNA and ssRNA in distinct sequence-dependent manners. While the helicase core plays an essential role in unwinding RNA/RNA and RNA/DNA duplexes, the N-terminal extension in HrpA, consisting of three helices referred to as the APHB domain, is crucial for ssDNA binding and RNA/DNA duplex unwinding. Importantly, the APHB domain is implicated in binding to non-canonical DNA structures such as G-quadruplex and i-motif, and this report presents the first solved i-motif-helicase complex. This research not only provides comprehensive insights into the multifaceted roles of HrpA as an RNA helicase but also establishes a foundation for further investigations into the recognition and functional implications of i-motif DNA structures in various biological processes.

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.

Nonstructural N- and C-tails of Dbp2 confer the protein full helicase activities.

Human DDX5 and its yeast ortholog Dbp2 are ATP-dependent RNA helicases that play a key role in normal cell processes, cancer development, and viral infection. The crystal structure of the RecA1-like domain of DDX5 is available but the global structure of DDX5/Dbp2 subfamily proteins remains to be elucidated. Here, we report the first X-ray crystal structures of the Dbp2 helicase core alone and in complex with ADP at 3.22 Å and 3.05 Å resolutions, respectively. The structures of the ADP-bound post-hydrolysis state and apo-state demonstrate the conformational changes that occur when the nucleotides are released. Our results showed that the helicase core of Dbp2 shifted between open and closed conformation in solution but the unwinding activity was hindered when the helicase core was restricted to a single conformation. A small-angle X-ray scattering experiment showed that the disordered amino (N) tail and carboxy (C) tails are flexible in solution. Truncation mutations confirmed that the terminal tails were critical for the nucleic acid binding, ATPase, and unwinding activities, with the C-tail being exclusively responsible for the annealing activity. Furthermore, we labeled the terminal tails to observe the conformational changes between the disordered tails and the helicase core upon binding nucleic acid substrates. Specifically, we found that the nonstructural terminal tails bind to RNA substrates and tether them to the helicase core domain, thereby conferring full helicase activities to the Dbp2 protein. This distinct structural characteristic provides new insight into the mechanism of DEAD-box RNA helicases.

Structural Studies of Pif1 Helicases from Thermophilic Bacteria.

Pif1 proteins are DNA helicases belonging to Superfamily 1, with 5' to 3' directionality. They are conserved from bacteria to human and have been shown to be particularly important in eukaryotes for replication and nuclear and mitochondrial genome stability. However, Pif1 functions in bacteria are less known. While most Pif1 from mesophilic bacteria consist of the helicase core with limited N-terminal and C-terminal extensions, some Pif1 from thermophilic bacteria exhibit a C-terminal WYL domain. We solved the crystal structures of Pif1 helicase cores from thermophilic bacteria Deferribacter desulfuricans and Sulfurihydrogenibium sp. in apo and nucleotide bound form. We show that the N-terminal part is important for ligand binding. The full-length Pif1 helicase was predicted based on the Alphafold algorithm and the nucleic acid binding on the Pif1 helicase core and the WYL domain was modelled based on known crystallographic structures. The model predicts that amino acids in the domains 1A, WYL, and linker between the Helicase core and WYL are important for nucleic acid binding. Therefore, the N-terminal and C-terminal extensions may be necessary to strengthen the binding of nucleic acid on these Pif1 helicases. This may be an adaptation to thermophilic conditions.

Biochemical and functional characterization of an exonuclease from Chaetomium thermophilum.

Exonucleases are often found associated with polymerase or helicase domains in the same enzyme or can function as autonomous entities to maintain genome stability. Here, we uncovered Chaetomium thermophilum RecQ family proteins that also have exonuclease activity in addition to their main helicase function. The novel exonuclease activity is separate from the helical core domain and coexists with the latter two enzymatic activities on the same polypeptide. The CtRecQ121-366 exonuclease region performs independently as an exonuclease. We describe its catalytic mechanism and biological characteristics. We demonstrate unequivocally that CtRecQ121-366 exclusively displays exonuclease activity and that this activity has a 3'-5' polarity that can both hydrolyze ssDNA and cleave dsDNA substrates. The hydrolytic activity of majority exonuclease is driven by bimetal ions, and this appears to be the case for the CtRecQ121-366 exonuclease as well. Additionally, the maximum activity of CtRecQ121-366 was observed at pH 8.0-9.0, low salt with Mg2+. The two helices in the structure, a6 and a7, play significant roles in the execution by anticipating their shape and changing essential amino acids.

DEAD-box RNA helicase Dbp2 binds to G-quadruplex nucleic acids and regulates different conformation of G-quadruplex DNA.

G-quadruplexes (G4s) are important in regulating DNA replication, repair and RNA transcription through interactions with specialized proteins. Dbp2 has been identified as a G4 DNA binding protein from Saccharomyces cerevisiae cell lysates. The majority of G4 motifs in Saccharomyces cerevisiae display 5-50 nt loops, only a few have 1-2 nt loops. Human DDX5 could unfold MycG4 DNA, whether Dbp2 also participates in remodeling G4 motifs with short loops in Saccharomyces cerevisiae remains elusive. Here we find that Dbp2 prefers G-rich substrates and binds MycG4 with a high affinity. Dbp2 possesses a dual function for different conformations of MycG4, destabilizing the folded MycG4 and inducing further folding of the unfolded MycG4. Similarly, DDX5 can unfold MycG4, but it exhibits a weaker MycG4 folding-promoting activity relative to Dbp2. Furthermore, Dbp2 facilitates DNA annealing activity in the absence of ATP, suggesting that Dbp2 can work on DNA substrates and possibly participate in DNA metabolism. Our results demonstrate that Dbp2 plays an important role in regulating the folding and unfolding activities of MycG4.

Recent advances in applying G-quadruplex for SARS-CoV-2 targeting and diagnosis: A review.

The coronavirus SARS-CoV-2 has caused a health care crisis all over the world since the end of 2019. Although vaccines and neutralizing antibodies have been developed, rapidly emerging variants usually display stronger immune escape ability and can better surpass vaccine protection. Therefore, it is still vital to find proper treatment strategies. To date, antiviral drugs against SARS-CoV-2 have mainly focused on proteases or polymerases. Notably, noncanonical nucleic acid structures called G-quadruplexes (G4s) have been identified in many viruses in recent years, and numerous G4 ligands have been developed. During this pandemic, literature on SARS-CoV-2 G4s is rapidly accumulating. Here, we first summarize the recent progress in the identification of SARS-CoV-2 G4s and their intervention by ligands. We then introduce the potential interacting proteins of SARS-CoV-2 G4s from both the virus and the host that may regulate G4 functions. The innovative strategy to use G4s as a diagnostic tool in SARS-CoV-2 detection is also reviewed. Finally, we discuss some key questions to be addressed in the future.

Bloom Syndrome Helicase Compresses Single-Stranded DNA into Phase-Separated Condensates.

Bloom syndrome protein (BLM) is a conserved RecQ family helicase involved in the maintenance of genome stability. BLM has been widely recognized as a genome "caretaker" that processes structured DNA. In contrast, our knowledge of how BLM behaves on single-stranded (ss) DNA is still limited. Here, we demonstrate that BLM possesses the intrinsic ability for phase separation and can co-phase separate with ssDNA to form dynamically arrested protein/ssDNA co-condensates. The introduction of ATP potentiates the capability of BLM to condense on ssDNA, which further promotes the compression of ssDNA against a resistive force of up to 60 piconewtons. Moreover, BLM is also capable of condensing replication protein A (RPA)- or RAD51-coated ssDNA, before which it generates naked ssDNA by dismantling these ssDNA-binding proteins. Overall, our findings identify an unexpected characteristic of a DNA helicase and provide a new angle of protein/ssDNA co-condensation for understanding the genomic instability caused by BLM overexpression under diseased conditions.

Targeting the RNA G-Quadruplex and Protein Interactome for Antiviral Therapy.

In recent years, G-quadruplexes (G4s), types of noncanonical four-stranded nucleic acid structures, have been identified in many viruses that threaten human health, such as HIV and Epstein-Barr virus. In this context, G4 ligands were designed to target the G4 structures, among which some have shown promising antiviral effects. In this Perspective, we first summarize the diversified roles of RNA G4s in different viruses. Next, we introduce small-molecule ligands developed as G4 modulators and highlight their applications in antiviral studies. In addition to G4s, we comprehensively review the medical intervention of G4-interacting proteins from both the virus (N protein, viral-encoded helicases, severe acute respiratory syndrome-unique domain, and Epstein-Barr nuclear antigen 1) and the host (heterogeneous nuclear ribonucleoproteins, RNA helicases, zinc-finger cellular nucelic acid-binding protein, and nucleolin) by inhibitors as an alternative way to disturb the normal functions of G4s. Finally, we discuss the challenges and opportunities in G4-based antiviral therapy.

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.

The convergence of head-on DNA unwinding forks induces helicase oligomerization and activity transition.

Helicases are multifunctional motor proteins with the primary task of separating nucleic acid duplexes. These enzymes often exist in distinct oligomeric forms and play essential roles during nucleic acid metabolism. Whether there is a correlation between their oligomeric state and cellular function, and how helicases effectively perform functional switching remains enigmatic. Here, we address these questions using a combined single-molecule approach and Bloom syndrome helicase (BLM). By examining the head-on collision of two BLM-mediated DNA unwinding forks, we find that two groups of BLM, upon fork convergence, promptly oligomerize across the fork junctions and tightly bridge two independent single-stranded (ss) DNA molecules that were newly generated by the unwinding BLMs. This protein oligomerization is mediated by the helicase and RNase D C-terminal (HRDC) domain of BLM and can sustain a disruptive force of up to 300 pN. Strikingly, onsite BLM oligomerization gives rise to an immediate transition of their helicase activities, from unwinding dsDNA to translocating along ssDNA at exceedingly fast rates, thus allowing for the efficient displacement of ssDNA-binding proteins, such as RPA and RAD51. These findings uncover an activity transition pathway for helicases and help to explain how BLM plays both pro- and anti-recombination roles in the maintenance of genome stability.

Remodeling the conformational dynamics of I-motif DNA by helicases in ATP-independent mode at acidic environment.

I-motifs are noncanonical four-stranded DNA structures formed by C-rich sequences at acidic environment with critical biofunctions. The particular pH sensitivity has inspired the development of i-motifs as pH sensors and DNA motors in nanotechnology. However, the folding and regulation mechanisms of i-motifs remain elusive. Here, using single-molecule FRET, we first show that i-motifs are more dynamic than G4s. Impressively, i-motifs display a high diversity of six folding species with slow interconversion. Further results indicate that i-motifs can be linearized by Replication protein A. More importantly, we identified a number of helicases with high specificity to i-motifs at low pH. All these helicases directly act on and efficiently resolve i-motifs into intermediates independent of ATP, although they poorly unwind G4 or duplex at low pH. Owing to the extreme sensitivity to helicases and no need for ATP, i-motif may be applied as a probe for helicase sensing both in vitro and in vivo.

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.

G-quadruplex DNA: a novel target for drug design.

G-quadruplex (G4) DNA is a type of quadruple helix structure formed by a continuous guanine-rich DNA sequence. Emerging evidence in recent years authenticated that G4 DNA structures exist both in cell-free and cellular systems, and function in different diseases, especially in various cancers, aging, neurological diseases, and have been considered novel promising targets for drug design. In this review, we summarize the detection method and the structure of G4, highlighting some non-canonical G4 DNA structures, such as G4 with a bulge, a vacancy, or a hairpin. Subsequently, the functions of G4 DNA in physiological processes are discussed, especially their regulation of DNA replication, transcription of disease-related genes (c-MYC, BCL-2, KRAS, c-KIT et al.), telomere maintenance, and epigenetic regulation. Typical G4 ligands that target promoters and telomeres for drug design are also reviewed, including ellipticine derivatives, quinoxaline analogs, telomestatin analogs, berberine derivatives, and CX-5461, which is currently in advanced phase I/II clinical trials for patients with hematologic cancer and BRCA1/2-deficient tumors. Furthermore, since the long-term stable existence of G4 DNA structures could result in genomic instability, we summarized the G4 unfolding mechanisms emerged recently by multiple G4-specific DNA helicases, such as Pif1, RecQ family helicases, FANCJ, and DHX36. This review aims to present a general overview of the field of G-quadruplex DNA that has progressed in recent years and provides potential strategies for drug design and disease treatment.

Replication protein A plays multifaceted roles complementary to specialized helicases in processing G-quadruplex DNA.

G-quadruplexes (G4s) are non-canonical DNA structures with critical roles in DNA metabolisms. To resolve those structures that can cause replication fork stalling and genomic instability, single-stranded DNA-binding proteins and helicases are required. Here, we characterized the interplay between RPA and helicases on G4s using single-molecule FRET. We first discovered that human RPA efficiently prevents G4 formation by preempting ssDNA before its folding. RPA also differentially interacts with the folded G4s. However, helicases such as human BLM and yeast Pif1 have different G4 preferences from RPA mainly based on loop lengths. More importantly, both RPA and these helicases are required for the stable G4 unfolding, as RPA promotes helicase-mediated repetitive unfolding into durative linear state. Furthermore, BLM can traverse G4 obstacles temporarily disrupted by RPA and continue to unwind downstream duplex. We finally proposed the mechanisms underlying above functions of RPA in preventing, resolving, and assisting helicases to eliminate G4s.

Structural study of the function of Candida Albicans Pif1.

Pif1 helicases, conserved in eukaryotes, are involved in maintaining genome stability in both the nucleus and mitochondria. Here, we report the crystal structure of a truncated Candida Albicans Pif1 (CaPif1368-883) in complex with ssDNA and an ATP analog. Our results show that the Q-motif is responsible for identifying adenine bases, and CaPif1 preferentially utilizes ATP/dATP during dsDNA unwinding. Although CaPif1 shares structural similarities with Saccharomyces cerevisiae Pif1, CaPif1 can contact the thymidine bases of DNA by hydrogen bonds, whereas ScPif1 cannot. More importantly, the crosslinking and mutant experiments have demonstrated that the conformational change of domain 2B is necessary for CaPif1 to unwind dsDNA. These findings contribute to further the understanding of the unwinding mechanism of Pif1.

Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi‡.

Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.

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.

Macromolecular aging: ATP hydrolysis-driven functional and structural changes in Escherichia coli RecQ helicase.

Aging has been considered a phenomenon that can be only applied to cells or organisms. Here, we show that RecQ helicase from E. coli displays an aging phenomenon: this macromolecular motor loses its structure and function after hydrolyzing a certain number of ATP molecules. The aging process was only triggered by repeated catalytic cycles. These observations lead to a new concept: macromolecule aging.