Metal-binding and folding thermodynamics of Escherichia coli ribonuclease HI related to its catalytic function.

Escherichia coli ribonuclease HI (RNH) hydrolyzes the RNA strands of RNA/DNA hybrids in the presence of Mg2+ at the highest level, relative to other metal ions. The Mg2+ binding affinity was 8.39 × 103 M-1, which was lower than those of other metal ions. The low-affinity binder can express the maximum catalytic activity of RNH. The stability of RNH increased with increasing metal ion concentration, except for Zn2+. The thermodynamic origin for enhancing the stability of RNH with Mg2+ was more favorable entropy compared to those with other metal ions, indicating that Mg2+ binding changes the RNH structure while maintaining flexibility. Upon H124A mutation, the metal ion binding affinities decreased for Mn2+ and Zn2+ to a relatively large extent. The present thermodynamic analyses provide information on the structural dynamics of RNH with metal ion exchangeable binding, which can reasonably explain the metal-ion-dependent catalytic activity.

Adjusting for publication bias in meta-analysis via inverse probability weighting using clinical trial registries.

Publication bias is a major concern in conducting systematic reviews and meta-analyses. Various sensitivity analysis or bias-correction methods have been developed based on selection models, and they have some advantages over the widely used trim-and-fill bias-correction method. However, likelihood methods based on selection models may have difficulty in obtaining precise estimates and reasonable confidence intervals, or require a rather complicated sensitivity analysis process. Herein, we develop a simple publication bias adjustment method by utilizing the information on conducted but still unpublished trials from clinical trial registries. We introduce an estimating equation for parameter estimation in the selection function by regarding the publication bias issue as a missing data problem under the missing not at random assumption. With the estimated selection function, we introduce the inverse probability weighting (IPW) method to estimate the overall mean across studies. Furthermore, the IPW versions of heterogeneity measures such as the between-study variance and the I2 measure are proposed. We propose methods to construct confidence intervals based on asymptotic normal approximation as well as on parametric bootstrap. Through numerical experiments, we observed that the estimators successfully eliminated bias, and the confidence intervals had empirical coverage probabilities close to the nominal level. On the other hand, the confidence interval based on asymptotic normal approximation is much wider in some scenarios than the bootstrap confidence interval. Therefore, the latter is recommended for practical use.

Pivotal role of a conserved histidine in Escherichia coli ribonuclease HI as proposed by X-ray crystallography.

The ribonuclease (RNase) H family of enzymes catalyze the specific cleavage of RNA strands of RNA/DNA hybrid duplexes and play an important role in DNA replication and repair. Since the first report of the crystal structure of RNase HI, its catalytic mechanisms, which require metal ions, have been discussed based on numerous structural and functional analyses, including X-ray crystallography. In contrast, the function of the conserved histidine residue (His124 in Escherichia coli) in the flexible loop around the active site remains poorly understood, although an important role was suggested by NMR analyses. Here, novel high-resolution X-ray crystal structures of E. coli RNase HI are described, with a particular focus on the interactions of divalent cations with His124 oriented towards the active site. The enzyme-Mg2+ complex contains two metal ions in the active site, one of which has previously been observed. The second ion lies alongside the first and binds to His124 in an octahedral coordination scheme. In the enzyme-Zn2+ complex a single metal ion was found to bind to the active site, showing a tetrahedral coordination geometry with the surrounding atoms, including His124. These results provide structural evidence that His124 plays a crucial role in the catalytic activity of RNase HI by interacting weakly and transiently with metal ions in the catalytic center.

G20 Summit and emergency medical services in Osaka, Japan.

AIM: To assess the impact of the Summit on Financial Markets and the World Economy held in Osaka City, Japan (G20 Osaka Summit) on the emergency medical services (EMS) system. METHODS: This study used the ORION database with its population-based registry of emergency patients comprising both ambulance and in-hospital records in Osaka Prefecture, Japan. The G20 Osaka Summit was held in Osaka City from 28 to 29 June, 2019. Changes in the EMS system and traffic regulations in Osaka were made during the period from 27 to 30 June, but we focused on the two summit days as the G20 period. The control periods comprised the same calendar days 1 week before and 1 week after the G20 period. We evaluated differences in the number of emergency transports, difficulties in obtaining hospital acceptance of patients, deaths among hospitalized emergency patients, and ambulance transport times between the two periods. RESULTS: In total, 2,590 cases in the G20 period and 5,152 cases in the control periods were registered. The relative risk of cases during the G20 versus control periods was 1.01 (0.96-1.05). Significant decreases were observed in the number of traffic accidents as ambulance calls (relative risk = 0.77; 95% confidence interval, 0.64-0.91). There were no significant differences in difficulties in obtaining hospital acceptance or deaths among hospitalized emergency patients between the G20 and control periods. In addition, ambulance transport times during the G20 period were not significantly longer than those in the control periods. CONCLUSION: The G20 Osaka Summit did not adversely impact the provision of emergency medical care in the Osaka area.

Identification of the ternary complex of ribonuclease HI:RNA/DNA hybrid:metal ions by ESI mass spectrometry.

Ribonuclease HI, an endoribonuclease, catalyzes the hydrolysis of the RNA strand of an RNA/DNA hybrid and requires divalent metal ions for its enzymatic activity. However, the mechanistic details of the activity of ribonuclease HI and its interaction with divalent metal ions remain unclear. In this study, we performed real-time monitoring of the enzyme-substrate complex in the presence of divalent metal ions (Mn2+ or Zn2+) using electrospray ionization-mass spectrometry (ESI-MS). The findings provide clear evidence that the enzymatic activity of the ternary complex requires the binding of two divalent metal ions. The Zn2+ ions bind to both the enzyme itself and the enzyme:substrate complex more strongly than Mn2+ ions, and gives, in part, the ternary complex, [RNase HI:nicked RNA/DNA hybrid:2Zn2+], suggesting that the ternary complex is retained, even after the hydrolysis of the substrate. The collective results presented herein shed new light on the essential role of divalent metal ions in the activity of ribonuclease HI and demonstrate how Zn2+ ions confer inhibitory properties on the activity of this enzyme by forming a highly stable complex with the substrate.

Partial Replacement of Nucleosomal DNA with Human FACT Induces Dynamic Exposure and Acetylation of Histone H3 N-Terminal Tails.

The FACT (facilitates chromatin transcription) complex, comprising SPT16 and SSRP1, conducts structural alterations during nucleosome unwrapping. Our previous cryoelectron microscopic (cryo-EM) analysis revealed the first intermediate structure of an unwrapped nucleosome with human FACT, in which 112-bp DNA and the phosphorylated intrinsically disordered (pAID) segment of SPT16 jointly wrapped around the histone core instead of 145-bp DNA. Using NMR, here we clarified that the histone H3 N-terminal tails, unobserved in the cryo-EM structure, adopt two different conformations reflecting their asymmetric locations at entry/exit sites: one corresponds to the original nucleosome site buried in two DNA gyres (DNA side), whereas the other, comprising pAID and DNA, is more exposed to the solvent (pAID side). NMR real-time monitoring showed that H3 acetylation is faster on the pAID side than on the DNA side. Our findings highlight that accessible conformations of H3 tails are created by the replacement of nucleosomal DNA with pAID.

Two conformations of DNA polymerase D-PCNA-DNA, an archaeal replisome complex, revealed by cryo-electron microscopy.

BACKGROUND: DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3'-5' editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. However, the processivity of the replicase holoenzyme has additionally been shown to be enhanced by the clamp molecule proliferating cell nuclear antigen (PCNA), making it crucial to elucidate the interaction between PolD and PCNA on a structural level for a full understanding of its functional relevance. We present here the 3D structure of a PolD-PCNA-DNA complex from Thermococcus kodakarensis using single-particle cryo-electron microscopy (EM). RESULTS: Two distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Pyrococcus abyssi onto our EM map showed the 3D atomic structural model of PolD-PCNA-DNA. In addition to the canonical interaction between PCNA and PolD via PIP (PCNA-interacting protein)-box motif, we found a new contact point consisting of a glutamate residue at position 171 in a ß-hairpin of PCNA, which mediates interactions with DP1 and DP2. The DNA synthesis activity of a mutant PolD with disruption of the E171-mediated PCNA interaction was not stimulated by PCNA in vitro. CONCLUSIONS: Based on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for conversion of its activity. This hook function of the clamp molecule may be conserved in the three domains of life.

Structural visualization of key steps in nucleosome reorganization by human FACT.

Facilitates chromatin transcription (FACT) is a histone chaperone, which accomplishes both nucleosome assembly and disassembly. Our combined cryo-electron microscopy (EM) and native mass spectrometry (MS) studies revealed novel key steps of nucleosome reorganization conducted by a Mid domain and its adjacent acidic AID segment of human FACT. We determined three cryo-EM structures of respective octasomes complexed with the Mid-AID and AID regions, and a hexasome alone. We discovered extensive contacts between a FACT region and histones H2A, H2B, and H3, suggesting that FACT is competent to direct functional replacement of a nucleosomal DNA end by its phosphorylated AID segment (pAID). Mutational assays revealed that the aromatic and phosphorylated residues within pAID are essential for octasome binding. The EM structure of the hexasome, generated by the addition of Mid-pAID or pAID, indicated that the dissociation of H2A-H2B dimer causes significant alteration from the canonical path of the nucleosomal DNA.

Full Moon and Out-of-Hospital Cardiac Arrest in Japan　- Population-Based, Double-Controlled Case Series Analysis.

Background: It is commonly believed that a full moon affects human behavior or the occurrence and outcome of various diseases; thus, the occurrence of out-of-hospital cardiac arrest (OHCA) might increase during full moon nights. Methods and Results: This nationwide, population-based observational study consecutively enrolled OHCA patients in Japan with attempted resuscitation between 2005 and 2016. The primary outcome measure was the occurrence of OHCA. Based on the double-control method, assuming Poisson sampling, we evaluated the average number of OHCA events that occurred on full moon nights compared with that which occurred on control nights, which included events that occurred on the same calendar days 1 week before and after the full moon nights. A total of 29,552 OHCA that occurred on 148 full moon nights and 58,707 OHCA that occurred on 296 control nights were eligible for analysis. The occurrence of OHCA did not differ between full moon and control nights (199.7 vs. 198.3 per night; relative risk [RR], 1.007; 95% CI: 0.993-1.021). On subgroup analysis, compared with control nights, the RR of OHCA occurrence were 1.013 (95% CI: 0.994-1.032, P=0.166) and 0.998 (95% CI: 0.977-1.020, P=0.866) for cardiac and non-cardiac origins, respectively. Conclusions: In this population, there was no significant difference in OHCA occurrence between full moon and control nights.

Direct visualization of DNA baton pass between replication factors bound to PCNA.

In Eukarya and Archaea, the lagging strand synthesis is accomplished mainly by three key factors, DNA polymerase (Pol), flap endonuclease (FEN), and DNA ligase (Lig), in the DNA replication process. These three factors form important complexes with proliferating cell nuclear antigen (PCNA), thereby constructing a platform that enable each protein factor to act successively and smoothly on DNA. The structures of the Pol-PCNA-DNA and Lig-PCNA-DNA complexes alone have been visualized by single particle analysis. However, the FEN-PCNA-DNA complex structure remains unknown. In this report, we for the first time present this tertiary structure determined by single particle analysis. We also successfully visualized the structure of the FEN-Lig-PCNA-DNA complex, corresponding to a putative intermediate state between the removal of the DNA flap by FEN and the sealing of the nicked DNA by Lig. This structural study presents the direct visualization of the handing-over action, which proceeds between different replication factors on a single PCNA clamp bound to DNA. We detected a drastic conversion of the DNA from a bent form to a straight form, in addition to the dynamic motions of replication factors in the switching process.

A Dual Base Flipping Mechanism for Archaeal Mismatch Repair.

In this issue of Structure, Nakae et al. (2016) report the structure of the archaeal EndoMS endonuclease, which cleaves DNA containing mismatched base pairs. Their data demonstrate a unique dual base flipping mechanism, providing intriguing insights into the molecular evolution of protein machineries involved in DNA mismatch repair.

Integrated molecular mechanism directing nucleosome reorganization by human FACT.

Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT-histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3-H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular ß structure with H4. At the other site, the Mid-H2A steric collision on the H2A-docking surface of the H3-H4 tetramer within the nucleosome induces H2A-H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone.

Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both ß-1,4-mannosidic and ß-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

Phosphorylation-coupled intramolecular dynamics of unstructured regions in chromatin remodeler FACT.

The intrinsically disordered region (IDR) of a protein is an important topic in molecular biology. The functional significance of IDRs typically involves gene-regulation processes and is closely related to posttranslational modifications such as phosphorylation. We previously reported that the Drosophila facilitates chromatin transcription (FACT) protein involved in chromatin remodeling contains an acidic ID fragment (AID) whose phosphorylation modulates FACT binding to nucleosomes. Here, we performed dynamic atomic force microscopy and NMR analyses to clarify how the densely phosphorylated AID masks the DNA binding interface of the high-mobility-group domain (HMG). Dynamic atomic force microscopy of the nearly intact FACT revealed that a small globule temporally appears but quickly vanishes within each mobile tail-like image, corresponding to the HMG-containing IDR. The lifespan of the globule increases upon phosphorylation. NMR analysis indicated that phosphorylation induces no ordered structure but increases the number of binding sites in AID to HMG with an adjacent basic segment, thereby retaining the robust electrostatic intramolecular interaction within FACT even in the presence of DNA. These data lead to the conclusion that the inhibitory effect of nucleosome binding is ascribed to the increase in the probability of encounter between HMG and the phosphorylated IDR.

Design and synthesis of a series of α-benzyl phenylpropanoic acid-type peroxisome proliferator-activated receptor (PPAR) gamma partial agonists with improved aqueous solubility.

In the continuing study directed toward the development of peroxisome proliferator-activated receptor gamma (hPPARγ) agonist, we attempted to improve the water solubility of our previously developed hPPARγ-selective agonist 3, which is insufficiently soluble for practical use, by employing two strategies: introducing substituents to reduce its molecular planarity and decreasing its hydrophobicity via replacement of the adamantyl group with a heteroaromatic ring. The first approach proved ineffective, but the second was productive. Here, we report the design and synthesis of a series of α-benzyl phenylpropanoic acid-type hPPARγ partial agonists with improved aqueous solubility. Among them, we selected (R)-7j, which activates hPPARγ to the extent of about 65% of the maximum observed with a full agonist, for further evaluation. The ligand-binding mode and the reason for the partial-agonistic activity are discussed based on X-ray-determined structure of the complex of hPPARγ ligand-binding domain (LBD) and (R)-7j with previously reported ligand-LDB structures. Preliminal apoptotic effect of (R)-7j against human scirrhous gastric cancer cell line OCUM-2MD3 is also described.

Structure-based mutational study of an archaeal DNA ligase towards improvement of ligation activity.

DNA ligases catalyze the joining of strand breaks in duplex DNA. The DNA ligase of Pyrococcus furiosus (PfuLig), which architecturally resembles the human DNA ligase I (hLigI), comprises an N-terminal DNA-binding domain, a middle adenylylation domain, and a C-terminal oligonucleotide-binding (OB)-fold domain. Here we addressed the C-terminal helix in the OB-fold domain of PfuLig by mutational analysis. The crystal structure of PfuLig revealed that this helix stabilizes a closed conformation of the enzyme by forming several ionic interactions with the adenylylation domain. The C-terminal helix is oriented differently in hLigI when DNA is bound; this suggested that disruption of its interaction with the adenylylation domain might facilitate the binding of DNA substrates. We indeed identified one of its residues, Asp540, as being critical for ligation efficiency. The D540R mutation improved the overall ligation activity relative to the wild-type enzyme, and at lower temperatures; this is relevant to applications such as ligation amplification reactions. Physical and biochemical analyses indicated that the improved ligation activity of the D540R variant arises from effects on the ligase adenylylation step and on substrate DNA binding in particular.

Bidirectional fluorescence properties of pyrene-based peroxisome proliferator-activated receptor (PPAR) α/δ dual agonist.

Based on X-ray crystallographic analysis of a peroxisome proliferator-activated receptor (PPAR) α/δ dual agonist complexed with human PPARs ligand binding domain (LBD), we previously reported the design and synthesis of a pyrene-based fluorescent PPARα/δ co-agonist 2. Here, we found that the fluorescence intensity of 2 increased upon binding to hPPARα-LBD, in a manner dependent upon the concentration of the LBD. But, surprisingly, the fluorescence intensity of 2 decreased concentration-dependently upon binding to hPPRδ-LBD. Site-directed mutagenesis of the two hPPAR subtypes clearly indicated that Trp264 of hPPARδ-LBD, located between H2' helix and H3 helix (omega loop), is critical for the concentration-dependent decrease in fluorescence intensity, which is suggested to be due to fluorescence resonance energy transfer (FRET) from the pyrene moiety of bound 2 to the nearby side-chain indole moiety of Trp264 in the hPPARδ-LBD.

CENP-T-W-S-X forms a unique centromeric chromatin structure with a histone-like fold.

The multiprotein kinetochore complex must assemble at a specific site on each chromosome to achieve accurate chromosome segregation. Defining the nature of the DNA-protein interactions that specify the position of the kinetochore and provide a scaffold for kinetochore formation remain key goals. Here, we demonstrate that the centromeric histone-fold-containing CENP-T-W and CENP-S-X complexes coassemble to form a stable CENP-T-W-S-X heterotetramer. High-resolution structural analysis of the individual complexes and the heterotetramer reveals similarity to other histone fold-containing complexes including canonical histones within a nucleosome. The CENP-T-W-S-X heterotetramer binds to and supercoils DNA. Mutants designed to compromise heterotetramerization or the DNA-protein contacts around the heterotetramer strongly reduce the DNA binding and supercoiling activities in vitro and compromise kinetochore assembly in vivo. These data suggest that the CENP-T-W-S-X complex forms a unique nucleosome-like structure to generate contacts with DNA, extending the "histone code" beyond canonical nucleosome proteins.

Architectures of archaeal GINS complexes, essential DNA replication initiation factors.

BACKGROUND: In the early stage of eukaryotic DNA replication, the template DNA is unwound by the MCM helicase, which is activated by forming a complex with the Cdc45 and GINS proteins. The eukaryotic GINS forms a heterotetramer, comprising four types of subunits. On the other hand, the archaeal GINS appears to be either a tetramer formed by two types of subunits in a 2:2 ratio (α2ß2) or a homotetramer of a single subunit (α4). Due to the low sequence similarity between the archaeal and eukaryotic GINS subunits, the atomic structures of the archaeal GINS complexes are attracting interest for comparisons of their subunit architectures and organization. RESULTS: We determined the crystal structure of the α2ß2 GINS tetramer from Thermococcus kodakaraensis (TkoGINS), comprising Gins51 and Gins23, and compared it with the reported human GINS structures. The backbone structure of each subunit and the tetrameric assembly are similar to those of human GINS. However, the location of the C-terminal small domain of Gins51 is remarkably different between the archaeal and human GINS structures. In addition, TkoGINS exhibits different subunit contacts from those in human GINS, as a consequence of the different relative locations and orientations between the domains. Based on the GINS crystal structures, we built a homology model of the putative homotetrameric GINS from Thermoplasma acidophilum (TacGINS). Importantly, we propose that a long insertion loop allows the differential positioning of the C-terminal domains and, as a consequence, exclusively leads to the formation of an asymmetric homotetramer rather than a symmetrical one. CONCLUSIONS: The DNA metabolizing proteins from archaea are similar to those from eukaryotes, and the archaeal multi-subunit complexes are occasionally simplified versions of the eukaryotic ones. The overall similarity in the architectures between the archaeal and eukaryotic GINS complexes suggests that the GINS function, directed through interactions with other protein components, is basically conserved. On the other hand, the different subunit contacts, including the locations and contributions of the C-terminal domains to the tetramer formation, imply the possibility that the archaeal and eukaryotic GINS complexes contribute to DNA unwinding reactions by significantly different mechanisms in terms of the atomic details.