ATPase Site Research Articles

The Role of SF1 and SF2 Helicases in Biotechnological Applications.

Helicases, which utilize ATP hydrolysis to separate nucleic acid duplexes, play crucial roles in DNA and RNA replication, repair, recombination, and transcription. Categorized into the major groups superfamily 1 (SF1) and superfamily 2 (SF2), alongside four minor groups, these proteins exhibit a conserved catalytic core indicative of a shared evolutionary origin while displaying functional diversity through interactions with various substrates. This review summarizes the structures, functions and mechanisms of SF1 and SF2 helicases, with an emphasis on conserved ATPase sites and RecA-like domains essential for their enzymatic and nucleic acid binding capabilities. It highlights the unique 1B and 2B domains in SF1 helicases and their impact on enzymatic activity. The DNA unwinding process is detailed, covering substrate recognition, ATP hydrolysis, and conformational changes, while addressing debates over the active form of UvrD helicase and post-unwinding dissociation. More importantly, this review discusses the biotechnological potential of helicases in emerging technologies such as nanopore sequencing, protein sequencing, and isothermal amplification, focusing on their use in pathogen detection, biosensor enhancement, and cancer treatment. As understanding deepens, innovative applications in genome editing, DNA sequencing, and synthetic biology are anticipated.

Signal Propagation in the ATPase Domain of Mycobacterium tuberculosis DNA Gyrase from Dynamical-Nonequilibrium Molecular Dynamics Simulations.

DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A2B2) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of M. tuberculosis DNA gyrase relative to those from other bacterial species.

Mechanism of DNA unwinding by MCM8-9 in complex with HROB

HROB promotes the MCM8-9 helicase in DNA damage response. To understand how HROB activates MCM8-9, we defined their interaction interface. We showed that HROB makes important yet transient contacts with both MCM8 and MCM9, and binds the MCM8-9 heterodimer with the highest affinity. MCM8-9-HROB prefer branched DNA structures, and display low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexamer that assembles from dimers on DNA in the presence of ATP. The hexamer involves two repeating protein-protein interfaces between the alternating MCM8 and MCM9 subunits. One of these interfaces is quite stable and forms an obligate heterodimer across which HROB binds. The other interface is labile and mediates hexamer assembly, independently of HROB. The ATPase site formed at the labile interface contributes disproportionally more to DNA unwinding than that at the stable interface. Here, we show that HROB promotes DNA unwinding downstream of MCM8-9 loading and ring formation on ssDNA.

Abstract 5898: Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells

Abstract Compared to normal hematopoietic cells, AML cells exhibit a heightened reliance on mitochondrial metabolism for survival and proliferation. To support this unique phenotype, AML cells increase import of nuclear-encoded mitochondrial proteins which must be properly folded by mitochondrial chaperones, proteases, and heat shock proteins. Failure to properly fold these precursors leads to protein aggregation, mitochondrial dysfunction, and cell death. To evaluate the reliance of AML cells on maintaining mitochondrial proteostasis, we assessed gene dependency datasets (eg: depmap.org) and identified the mitochondrial protease LONP1, as the top essential gene. LONP1 is a matrix-localized nuclear-encoded AAA+ protease. Proteins are unfolded by its ATPase domain and degraded by its serine-catalyzed proteolytic domain. Compared to hematopoietic cells, LONP1 mRNA was overexpressed in AML across three publicly available datasets. By immunoblotting, LONP1 protein was increased in 16/30 primary AML samples compared to bulk (n=8) and CD34+ blood cells (n=3). LONP1 genetic depletion reduced the growth and viability of OCI-AML2, OCI-M2, NB4, and TEX leukemia cells as well as primary AML cells. LONP1 depletion increased aggregated mitochondrial proteins and reactive oxygen species (ROS) production while reducing mitochondrial respiration and membrane potential. To determine the domain of LONP1 that was necessary for mitochondrial protein solubility, mitochondrial function, and AML survival, we over-expressed wild type, ATPase dead (E591A), or proteolytically dead (S855A) LONP1 cDNA in OCI-AML2 cells and knocked down endogenous LONP1 with shRNA targeting the 3’UTR of the endogenous gene. Wild type or proteolytically dead (S855A) but not the ATPase mutant (E591A) rescued mitochondrial protein solubility, mitochondrial respiration, ROS generation, membrane potential, and cell viability. Thus, the ATPase domain but not the proteolytic activity of LONP1 is necessary for mitochondrial proteostasis. Bardoxolone methyl (CDDO-Me) is a synthetic triterpenoid that allosterically inhibits the LONP1 ATPase site. CDDO-Me killed OCI-AML2 and NB4 cells with IC50 values of 178.5±29.7 and 156.5±39.7 nM, respectively. CDDO-Me (200 nM) killed 3 out of 4 primary AML samples with high LONP1 expression but none of the 10 primary AML samples with low LONP1. Likewise, CDDO-Me induced mitochondrial protein aggregation in AML cell lines and primary AML patient samples with high LONP1 expression but not AML cells or primary AML patient samples with low LONP1. In summary, LONP1 is over-expressed in a subset of AML cells and primary samples where it maintains mitochondrial protein solubility. Selective inhibition of the LONP1 ATPase domain leads to mitochondrial protein aggregation and AML cell death, representing a novel therapeutic strategy for AML. Citation Format: Matthew Tcheng, Marcela Gronda, Rose Hurren, Lan Xin Zhang, Chaitra Sarathy, Yongran Yan, Andrea Arruda, Mark D. Minden, Aaron D. Schimmer. Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5898.

Identification of a novel DNA repair inhibitor using an insilico driven approach shows effective combinatorial activity with genotoxic agents against multidrug-resistant Escherichia coli.

Increasing antimicrobial drug resistance represents a global existential threat. Infection is a particular problem in immunocompromised individuals, such as patients undergoing cancer chemotherapy, due to the targeting of rapidly dividing cells by antineoplastic agents. We recently developed a strategy that targets bacterial nucleotide excision DNA repair (NER) to identify compounds that act as antimicrobial sensitizers specific for patients undergoing cancer chemotherapy. Building on this, we performed a virtual drug screening of a ~120,000 compound library against the key NER protein UvrA. From this, numerous target compounds were identified and of those a candidate compound, Bemcentinib (R428), showed a strong affinity toward UvrA. This NER protein possesses four ATPase sites in its dimeric state, and we found that Bemcentinib could inhibit UvrA's ATPase activity by ~90% and also impair its ability to bind DNA. As a result, Bemcentinib strongly diminishes NER's ability to repair DNA in vitro. To provide a measure of in vivo activity we discovered that the growth of Escherichia coli MG1655 was significantly inhibited when Bemcentinib was combined with the DNA damaging agent 4-NQO, which is analogous to UV. Using the clinically relevant DNA-damaging antineoplastic cisplatin in combination with Bemcentinib against the urological sepsis-causing E. coli strain EC958 caused complete growth inhibition. This study offers a novel approach for the potential development of new compounds for use as adjuvants in antineoplastic therapy.

Comparing ATPase activity of ATP-binding cassette subfamily C member 4, lamprey CFTR, and human CFTR using an antimony-phosphomolybdate assay.

Introduction: ATP-binding cassette (ABC) transporters use the hydrolysis of ATP to power the active transport of molecules, but paradoxically the cystic fibrosis transmembrane regulator (CFTR, ABCC7) forms an ion channel. We previously showed that ATP-binding cassette subfamily C member 4 (ABCC4) is the closest mammalian paralog to CFTR, compared to other ABC transporters. In addition, Lamprey CFTR (Lp-CFTR) is the oldest known CFTR ortholog and has unique structural and functional features compared to human CFTR (hCFTR). The availability of these evolutionarily distant orthologs gives us the opportunity to study the changes in ATPase activity that may be related to their disparate functions. Methods: We utilized the baculovirus expression system with Sf9 insect cells and made use of the highly sensitive antimony-phosphomolybdate assay for testing the ATPase activity of human ABCC4 (hABCC4), Lp-CFTR, and hCFTR under similar experimental conditions. This assay measures the production of inorganic phosphate (Pi) in the nanomolar range. Results: Crude plasma membranes were purified, and protein concentration, determined semi-quantitatively, of hABCC4, Lp-CFTR, and hCFTR ranged from 0.01 to 0.36μg/μL. No significant difference in expression level was found although hABCC4 trended toward the highest level. hABCC4 was activated by ATP with the equilibrium constant (Kd) 0.55 ± 0.28mM (n = 8). Estimated maximum ATPase rate (Vmax) for hABCC4 was about 0.2nmol/μg/min when the protein was activated with 1mM ATP at 37°C (n = 7). Estimated maximum ATPase rate for PKA-phosphorylated Lp-CFTR reached about half of hCFTR levels in the same conditions. Vmax for both Lp-CFTR and hCFTR were significantly increased in high PKA conditions compared to low PKA conditions. Maximum intrinsic ATPase rate of hABCC4 in the absence of substrate was twice that of hCFTR when activated in 1mM ATP. Conclusion: The findings here suggest that while both ABCC4 and hCFTR bear one consensus and one degenerate ATPase site, the hCFTR exhibited a reduced intrinsic ATPase activity. In addition, ATPase activity in the CFTR lineage increased from Lp-CFTR to hCFTR. Finally, the studies pave the way to purify hABCC4, Lp-CFTR, and hCFTR from Sf9 cells for their structural investigation, including by cryo-EM, and for studies of evolution in the ABC transporter superfamily.

Chi hotspot Control of RecBCD Helicase-nuclease: Enzymatic Tests Support the Intramolecular Signal-transduction Model

Repair of broken DNA is essential for life; the reactions involved can also promote genetic recombination to aid evolution. In Escherichia coli, RecBCD enzyme is required for the major pathway of these events. RecBCD is a complex ATP-dependent DNA helicase with nuclease activity controlled by Chi recombination hotspots (5′-GCTGGTGG-3′). During rapid DNA unwinding, when Chi is in a RecC tunnel, RecB nuclease nicks DNA at Chi. Here, we test our signal transduction model – upon binding Chi (step 1), RecC signals RecD helicase to stop unwinding (step 2); RecD then signals RecB (step 3) to nick at Chi (step 4) and to begin loading RecA DNA strand-exchange protein (step 5). We discovered that ATP-γ-S, like the small molecule RecBCD inhibitor NSAC1003, causes RecBCD to nick DNA, independent of Chi, at novel positions determined by the DNA substrate length. Two RecB ATPase-site mutants nick at novel positions determined by their RecB:RecD helicase rate ratios. In each case, we find that nicking at the novel position requires steps 3 and 4 but not step 1 or 2, as shown by mutants altered at the intersubunit contacts specific for each step; nicking also requires RecD helicase and RecB nuclease activities. Thus, altering the RecB ATPase site, by small molecules or mutation, sensitizes RecD to signal RecB to nick DNA (steps 4 and 3, respecitvely) without the signal from RecC or Chi (steps 1 and 2). These new, enzymatic results strongly support the signal transduction model and provide a paradigm for studying other complex enzymes.

Synergism between CMG helicase and leading strand DNA polymerase at replication fork

The replisome that replicates the eukaryotic genome consists of at least three engines: the Cdc45-MCM-GINS (CMG) helicase that separates duplex DNA at the replication fork and two DNA polymerases, one on each strand, that replicate the unwound DNA. Here, we determined a series of cryo-electron microscopy structures of a yeast replisome comprising CMG, leading-strand polymerase Polε and three accessory factors on a forked DNA. In these structures, Polε engages or disengages with the motor domains of the CMG by occupying two alternative positions, which closely correlate with the rotational movement of the single-stranded DNA around the MCM pore. During this process, the polymerase remains stably coupled to the helicase using Psf1 as a hinge. This synergism is modulated by a concerted rearrangement of ATPase sites to drive DNA translocation. The Polε-MCM coupling is not only required for CMG formation to initiate DNA replication but also facilitates the leading-strand DNA synthesis mediated by Polε. Our study elucidates a mechanism intrinsic to the replisome that coordinates the activities of CMG and Polε to negotiate any roadblocks, DNA damage, and epigenetic marks encountered during translocation along replication forks.

Abstract 4834: Mitochondrial ClpXP degrades serine phosphorylated protein aggregates

Abstract ClpXP is a serine protease localized to the mitochondrial matrix and maintains mitochondrial proteostasis by degrading damaged/aggregated respiratory chain complex proteins. Both inhibition and hyperactivation of ClpP lead to impaired OXPHOS function and kill leukemic cells in vitro and in vivo. Yet, the marks that tag proteins for degradation by ClpXP remain unknown. The bacterial homologue of ClpXP degrades damaged proteins with arginine phosphorylation. Therefore, we assessed how phosphorylation affects ClpXP protease activity. Recombinant ClpXP was incubated with its unnatural substrate, FITC-tagged casein, in the presence of increasing concentrations of phospho-serine (pSer), phospho-threonine (pThr), phospho-arginine (pArg), or phospho-tyrosine (pTyr). In a dose-dependent manner, pSer and pThr free amino acids and short peptides inhibited casein degradation by ClpXP. In contrast, free Ser, Thr, phosphate, pArg and pTyr did not inhibit ClpXP protease activity. In addition, we characterized ClpP peptidase activity and ClpX ATPase activity using the Ac-Trp-Leu-Ala-AMC (Ac-WLA-AMC) peptidase activity assay and the Malachite Green ATPase assay. Neither pSer nor pThr inhibited ClpX ATPase activity or ClpP peptidase activity. Thus, pSer and pThr only blocked ClpXP from degrading full-length protein without altering the enzymatic activity of the complex. Next, we characterized pSer and pThr binding to ClpXP by thermal shift binding assay. pSer and pThr free amino acids and short peptides bound to ClpX but not ClpP. All the dephosphorylated counterparts did not bind to ClpX. Therefore, pSer and pThr interact with ClpX outside the ATPase site. cyroEM studies of ClpX and pSer peptides indicated a putative pSer docking site in the central pore of ClpX. Previously, we identified OXPHOS complex II subunit SDHA as a potential ClpXP endogenous substrate. Therefore, we used SDHA as a model to assess whether serine phosphorylation influenced ClpXP protein degradation in intact cells. Using pSer immunoprecipitation, we observed increased serine phosphorylated SDHA in AML cells after ClpP and ClpX knockdown. The increased serine phosphorylated SDHA was specifically found in the insoluble fraction of the mitochondrial proteins. Supporting that serine phosphorylated SDHA represents aggregated protein, treatment of cells with ROS-inducing antimycin or 42°C heat shock also increased levels of serine phosphorylated SDHA in the fraction of insoluble mitochondrial proteins. Finally, adding recombinant ClpXP to mitochondrial lysates specifically degraded SDHA with serine phosphorylation. In summary, mitochondrial ClpXP degrades aggregated mitochondrial proteins marked by serine phosphorylated. Thus, this work identifies new degradation machinery for mitochondrial protease, advances the function of mitochondrial ClpXP, and highlights a new therapeutic strategy to target this protease. Citation Format: Yue Feng, Yulia Jitkova, Alexander Keszei, Jonathan St-Germain, Yongran Yan, Brian Raught, Mohammad Mazhab-Jafari, Aaron Schimmer. Mitochondrial ClpXP degrades serine phosphorylated protein aggregates. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4834.

Abstract 1906: MSH3: An underestimated DNA MMR gene in colorectal carcinogenesis and its potential role in disparity

Abstract Background: Unique mutations in cancer genes have been found in different races including African Americans (AA) population. Maintenance of proficient mismatch repair genes is important in colorectal cancer. Among them, MSH3 is found to be more important in colorectal cancer in AA particularly as its alterations associate with EMAST phenotype that has poor prognosis. However, there are regional variations in the classification and criteria used to determine the pathogenicity of these mutations particularly in AA due to their high genomic diversity. Aim: To analyze unique and novel MSH3 mutations using specific in-silico prediction tools and to validate their biological significances in AA-CRC. Results: In this study, we validated our targeted exome sequencing data of MSH3 mutations in AA and Caucasians (CA) CRC samples. A cost effective in-silico approach was employed to narrow down most significant MSH3 mutations. Interestingly, we and found six non-synonymous novel, pathogenic mutations in MSH3 gene (c.G1237A, c.C2759T, c.G1397A, c.G2926A, c.C3028T, c.G3241A) correspond to MSH3 protein (E413K; S466N; S920F; E976K; H1010Y; E1081K), in AA than CA samples. Also, we observed loss or gain of different chemical bonding patterns (hydrogen, ionic, hydrophobic, and di-sulfide bonding) between wild type and mutant MSH3 protein. Interestingly, some of these mutations were within the ATPase site while others are in MSH3-MSH2 interacting domains. Furthermore, Molecular Dynamics Simulation (MDS) of these six mutants of MSH3 proteins showed an overall effect on MSH3 protein stability especially in MSH3-MSH2 interaction compared to wild type MSH3 protein.Conclusion: Our data showed that, six MSH3 non-synonymous mutations are deleterious as they affect important sites within MSH3-MSH2 interactive and ATPase domain. Assessing the real frequency of these mutations in large cohort and their specific effect on function may shed light on EMAST phenotype, high prevalence, and overall poor prognosis in the AA population. In vitro functional assays using CRISPR-Cas9 knock-in and in vivo studies are underway to mimic these mutations. Citation Format: Mudasir Rashid, Rumaisa Rashid, Minoru Koi, John M. Carethers, Hassan Brim, Hassan Ashktorab. MSH3: An underestimated DNA MMR gene in colorectal carcinogenesis and its potential role in disparity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1906.

CFTR function, pathology and pharmacology at single-molecule resolution

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates salt and fluid homeostasis across epithelial membranes1. Alterations in CFTR cause cystic fibrosis, a fatal disease without a cure2,3. Electrophysiological properties of CFTR have been analysed for decades4–6. The structure of CFTR, determined in two globally distinct conformations, underscores its evolutionary relationship with other ATP-binding cassette transporters. However, direct correlations between the essential functions of CFTR and extant structures are lacking at present. Here we combine ensemble functional measurements, single-molecule fluorescence resonance energy transfer, electrophysiology and kinetic simulations to show that the two nucleotide-binding domains (NBDs) of human CFTR dimerize before channel opening. CFTR exhibits an allosteric gating mechanism in which conformational changes within the NBD-dimerized channel, governed by ATP hydrolysis, regulate chloride conductance. The potentiators ivacaftor and GLPG1837 enhance channel activity by increasing pore opening while NBDs are dimerized. Disease-causing substitutions proximal (G551D) or distal (L927P) to the ATPase site both reduce the efficiency of NBD dimerization. These findings collectively enable the framing of a gating mechanism that informs on the search for more efficacious clinical therapies.

Intramolecular tension and catch bonds play key roles in the conformational cycle of the dynein-like AAA mechanoenzyme Mdn1.

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically extract the assembly factors Rsa4 and Ytm1 from the pre-60S subunit during ribosome biogenesis. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with various activities) ring, which like dynein has six ATPase sites. The AAA ring is followed by large (>2000 amino acid) linking domains including an ∼500 amino acid disordered (D/E-rich) region, and a C-terminal assembly factor-binding MIDAS domain. Previous structural studies have revealed that when Mdn1 binds to the pre-60S, it docks its MIDAS domain onto its AAA ring and forms a tripartite connection between these two domains and the assembly factor that it is primed to extract. However, it is currently not known how this conformational state is formed and regulated. Here, we use a combination of optical tweezers, bulk biochemistry, and chemical biology approaches to investigate domain interactions and conformational changes in the Mdn1 mechanochemical cycle. We find that the MIDAS domain can bind stably to the AAA ring when they are separated into two different proteins, but that intramolecular tension built up along the linking domains prohibits this interaction in the full-length protein. We further find that the MIDAS domain can form catch bonds - bonds where tension enhances binding affinity - with both Rsa4 and Ytm1. Together, these results suggest a mechanoregulatory mechanism wherein intramolecular tension prevents MIDAS-AAA docking when Mdn1 is off the pre-60S, yet promotes the docked conformation when Mdn1 is on the pre-60S by activating the catch bond.

MDA5-dependent responses contribute to autoimmune diabetes progression and hindrance

Type 1 diabetes (T1D) is an autoimmune disease resulting in pancreatic β cell destruction. Coxsackievirus B3 (CVB3) infection and melanoma differentiation-associated protein 5-dependent (MDA5-dependent) antiviral responses are linked with T1D development. Mutations within IFIH1, coding for MDA5, are correlated with T1D susceptibility, but how these mutations contribute to T1D remains unclear. Utilizing nonobese diabetic (NOD) mice lacking Ifih1 expression (KO) or containing an in-frame deletion within the ATPase site of the helicase 1 domain of MDA5 (ΔHel1), we tested the hypothesis that partial or complete loss-of-function mutations in MDA5 would delay T1D by impairing proinflammatory pancreatic macrophage and T cell responses. Spontaneous T1D developed in female NOD and KO mice similarly, but was significantly delayed in ΔHel1 mice, which may be partly due to a concomitant increase in myeloid-derived suppressor cells. Interestingly, KO male mice had increased spontaneous T1D compared with NOD mice. Whereas NOD and KO mice developed CVB3-accelerated T1D, ΔHel1 mice were protected partly due to decreased type I IFNs, pancreatic infiltrating TNF+ macrophages, IFN-γ+CD4+ T cells, and perforin+CD8+ T cells. Furthermore, ΔHel1 MDA5 protein had reduced ATP hydrolysis compared with wild-type MDA5. Our results suggest that dampened MDA5 function delays T1D, yet loss of MDA5 promotes T1D.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Origin recognition complex harbors an intrinsic nucleosome remodeling activity

Eukaryotic DNA replication is initiated at multiple chromosomal sites known as origins of replication that are specifically recognized by the origin recognition complex (ORC) containing multiple ATPase sites. In budding yeast, ORC binds to specific DNA sequences known as autonomously replicating sequences (ARSs) that are mostly nucleosome depleted. However, nucleosomes may still inhibit the licensing of some origins by occluding ORC binding and subsequent MCM helicase loading. Using purified proteins and single-molecule visualization, we find here that the ORC can eject histones from a nucleosome in an ATP-dependent manner. The ORC selectively evicts H2A-H2B dimers but leaves the (H3-H4)2 tetramer on DNA. It also discriminates canonical H2A from the H2A.Z variant, evicting the former while retaining the latter. Finally, the bromo-adjacent homology (BAH) domain of the Orc1 subunit is essential for ORC-mediated histone eviction. These findings suggest that the ORC is a bona fide nucleosome remodeler that functions to create a local chromatin environment optimal for origin activity.

Molecular basis for polysaccharide recognition and modulated ATP hydrolysis by the O antigen ABC transporter

O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.

Mechanistic insight into the RNA-stimulated ATPase activity of tick-borne encephalitis virus helicase

The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase.

Ino80 is required for H2A.Z eviction from hypha-specific promoters and hyphal development of Candida albicans.

ATP‐dependent chromatin remodeling complexes play important roles in many essential cellular processes, including transcription regulation, DNA replication, and repair. Evicting H2A.Z, a variant of histone H2A, from the promoter of hypha‐specific genes is required for hyphal formation in Candida albicans. However, the mechanism that regulates H2A.Z removal during hyphal formation remains unknown. In this study, we demonstrated that Ino80, the core catalytic subunit of the INO80 complex, was recruited to hypha‐specific promoters during hyphal induction in Arp8 dependent manner and facilitated the removal of H2A.Z. Deleting INO80 or mutating the ATPase site of Ino80 impairs the expression of hypha‐specific genes (HSGs) and hyphal development. In addition, we showed that Ino80 was essential for the virulence of C. albicans during systemic infections in mice. Interestingly, Arp5, an INO80 complex‐specific component, acts in concert with Ino80 during DNA damage responses but is dispensable for hyphal induction. Our findings clarified that Ino80 was critical for hyphal development, DNA damage response, and pathogenesis in C. albicans.

Structural and functional insights into the mechanism by which MutS2 recognizes a DNA junction

MutS family proteins are classified into MutS-I and -II lineages: MutS-I recognizes mismatched DNA and initiates mismatch repair, whereas MutS-II recognizes DNA junctions to modulate recombination. MutS-I forms dimeric clamp-like structures enclosing the mismatched DNA, and its composite ATPase sites regulate DNA-binding modes. Meanwhile, the structures of MutS-II have not been determined; accordingly, it remains unknown how MutS-II recognizes DNA junctions and how nucleotides control DNA binding. Here, we solved the ligand-free and ADP-bound crystal structures of bacterial MutS2 belonging to MutS-II. MutS2 also formed a dimeric clamp-like structure with composite ATPase sites. The ADP-bound MutS2 was more flexible compared to the ligand-free form and could be more suitable for DNA entry. The inner hole of the MutS2 clamp was two times larger than that of MutS-I, and site-directed mutagenesis analyses revealed DNA-binding sites at the inner hole. Based on these, a model is proposed that describes how MutS2 recognizes DNA junctions.

A mechanism of origin licensing control through autoinhibition of S. cerevisiae ORC\xb7DNA\xb7Cdc6

The coordinated action of multiple replicative helicase loading factors is needed for the licensing of replication origins prior to DNA replication. Binding of the Origin Recognition Complex (ORC) to DNA initiates the ATP-dependent recruitment of Cdc6, Cdt1 and Mcm2-7 loading, but the structural details for timely ATPase site regulation and for how loading can be impeded by inhibitory signals, such as cyclin-dependent kinase phosphorylation, are unknown. Using cryo-electron microscopy, we have determined several structures of S. cerevisiae ORC·DNA·Cdc6 intermediates at 2.5–2.7 Å resolution. These structures reveal distinct ring conformations of the initiator·co-loader assembly and inactive ATPase site configurations for ORC and Cdc6. The Orc6 N-terminal domain laterally engages the ORC·Cdc6 ring in a manner that is incompatible with productive Mcm2-7 docking, while deletion of this Orc6 region alleviates the CDK-mediated inhibition of Mcm7 recruitment. Our findings support a model in which Orc6 promotes the assembly of an autoinhibited ORC·DNA·Cdc6 intermediate to block origin licensing in response to CDK phosphorylation and to avert DNA re-replication.