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.

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.

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.

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.

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.

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.

Structural analysis reveals a "molecular calipers" mechanism for a LATERAL ORGAN BOUNDARIES DOMAIN transcription factor protein from wheat.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins, a family of plant-specific transcription factors harboring a conserved Lateral Organ Boundaries (LOB) domain, are regulators of plant organ development. Recent studies have unraveled additional pivotal roles of the LBD protein family beyond defining lateral organ boundaries, such as pollen development and nitrogen metabolism. The structural basis for the molecular network of LBD-dependent processes remains to be deciphered. Here, we solved the first structure of the homodimeric LOB domain of Ramosa2 from wheat (TtRa2LD) to 1.9 Å resolution. Our crystal structure reveals structural features shared with other zinc-finger transcriptional factors, as well as some features unique to LBD proteins. Formation of the TtRa2LD homodimer relied on hydrophobic interactions of its coiled-coil motifs. Several specific motifs/domains of the LBD protein were also involved in maintaining its overall conformation. The intricate assembly within and between the monomers determined the precise spatial configuration of the two zinc fingers that recognize palindromic DNA sequences. Biochemical, molecular modeling, and small-angle X-ray scattering experiments indicated that dimerization is important for cooperative DNA binding and discrimination of palindromic DNA through a molecular calipers mechanism. Along with previously published data, this study enables us to establish an atomic-scale mechanistic model for LBD proteins as transcriptional regulators in plants.

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.

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.

Purification and enzymatic characterization of Gallus gallus BLM helicase.

Mutations in human BLM helicase give rise to the autosomal recessive Bloom syndrome, which shows high predisposition to types of malignant tumours. Though lots of biochemical and structural investigations have shed lights on the helicase core, structural investigations of the whole BLM protein are still limited due to its low stability and production. Here by comparing with the expression systems and functions of other BLM homologues, we developed the heterologous high-level expression and high-yield purification systems for Gallus gallus BLM (gBLM) in Escherichia coli. Subsequent DNA binding and unwinding determinations demonstrated that gBLM was a vigorous atypical DNA structure specific helicase, which not only showed high preference for the 3'-tailed DNA structures but also could efficiently unwind bubble DNA structures with blunt-ends, indicating its biological roles in processing DNA metabolism intermediates. Further comparative analysis between gBLM and gBLM Core revealed that the long N-terminal domain facilitated the binding affinity of forked and bubble DNA structures and it was also required for the DNA unwinding activities of gBLM. Thus, we present the first enzymatic characterization of gBLM and its N-terminal domain, providing a new model for probing the mechanism and structure of human BLM.

A helical bundle in the N-terminal domain of the BLM helicase mediates dimer and potentially hexamer formation.

Helicases play a critical role in processes such as replication or recombination by unwinding double-stranded DNA; mutations of these genes can therefore have devastating biological consequences. In humans, mutations in genes of three members of the RecQ family helicases (blm, wrn, and recq4) give rise to three strikingly distinctive clinical phenotypes: Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome, respectively. However, the molecular basis for these varying phenotypic outcomes is unclear, in part because a full mechanistic description of helicase activity is lacking. Because the helicase core domains are highly conserved, it has been postulated that functional differences among family members might be explained by significant differences in the N-terminal domains, but these domains are poorly characterized. To help fill this gap, we now describe bioinformatics, biochemical, and structural data for three vertebrate BLM proteins. We pair high resolution crystal structures with SAXS analysis to describe an internal, highly conserved sequence we term the dimerization helical bundle in N-terminal domain (DHBN). We show that, despite the N-terminal domain being loosely structured and potentially lacking a defined three-dimensional structure in general, the DHBN exists as a dimeric structure required for higher order oligomer assembly. Interestingly, the unwinding amplitude and rate decrease as BLM is assembled from dimer into hexamer, and also, the stable DHBN dimer can be dissociated upon ATP hydrolysis. Thus, the structural and biochemical characterizations of N-terminal domains will provide new insights into how the N-terminal domain affects the structural and functional organization of the full BLM molecule.

A 3'-5' exonuclease activity embedded in the helicase core domain of Candida albicans Pif1 helicase.

3'-5' exonucleases are frequently found to be associated to polymerases or helicases domains in the same enzyme or could function as autonomous entities. Here we uncovered that Candida albicans Pif1 (CaPif1) displays a 3'-5' exonuclease activity besides its main helicase activity. These two latter activities appear to reside on the same polypeptide and the new exonuclease activity could be mapped to the helicase core domain. We clearly show that CaPif1 displays exclusively exonuclease activity and unambiguously establish the directionality of the exonuclease activity as the 3'-to-5' polarity. The enzyme appears to follow the two-metal-ion driven hydrolyzing activity exhibited by most of the nucleases, as shown by its dependence of magnesium and also by the identification of aspartic residues. Interestingly, an excellent correlation could be found between the presence of the conserved residues and the exonuclease activity when testing activities on Pif1 enzymes from eight fungal organisms. In contrast to others proteins endowed with the double helicase/exonuclease functionality, CaPif1 differs in the fact that the two activities are embedded in the same helicase domain and not located on separated domains. Our findings may suggest a biochemical basis for mechanistic studies of Pif1 family helicases.

G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction.

Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a 'waiting time'. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells.

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.

The Bacteroides sp. 3_1_23 Pif1 protein is a multifunctional helicase.

ScPif1 DNA helicase is the prototypical member of a 5'-to-3' helicase superfamily conserved from bacteria to human and plays various roles in the maintenance of genomic homeostasis. While many studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Here, we report the expression, purification and biochemical analysis of Pif1 helicase from Bacteroides sp. 3_1_23 (BsPif1). BsPif1 binds to a large panel of DNA substrates and, in particular, efficiently unwinds partial duplex DNAs with 5'-overhang, fork-like substrates, D-loop and flap-like substrates, suggesting that BsPif1 may act at stalled DNA replication forks and enhance Okazaki fragment maturation. Like its eukaryotic homologues, BsPif1 resolves R-loop structures and unwinds DNA-RNA hybrids. Furthermore, BsPif1 efficiently unfolds G-quadruplexes and disrupts nucleoprotein complexes. Altogether, these results highlight that prokaryotic Pif1 helicases may resolve common issues that arise during DNA transactions. Interestingly, we found that BsPif1 is different from yeast Pif1, but resembles more human Pif1 with regard to substrate specificity, helicase activity and mode of action. These findings are discussed in the context of the possible functions of prokaryotic Pif1 helicases in vivo.