DNA glycosylases are important repair enzymes that eliminate a diverse array of aberrant nucleobases from the genomes of all organisms. Individual bacterial species often contain multiple paralogs of ...a particular glycosylase, yet the molecular and functional distinctions between these paralogs are not well understood. The recently discovered HEAT-like repeat (HLR) DNA glycosylases are distributed across all domains of life and are distinct in their specificity for cationic alkylpurines and mechanism of damage recognition. Here, we describe a number of phylogenetically diverse bacterial species with two orthologs of the HLR DNA glycosylase AlkD. One ortholog, which we designate AlkD2, is substantially less conserved. The crystal structure of Streptococcus mutans AlkD2 is remarkably similar to AlkD but lacks the only helix present in AlkD that penetrates the DNA minor groove. We show that AlkD2 possesses only weak DNA binding affinity and lacks alkylpurine excision activity. Mutational analysis of residues along this DNA binding helix in AlkD substantially reduced binding affinity for damaged DNA, for the first time revealing the importance of this structural motif for damage recognition by HLR glycosylases.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA methylation is one of the most important epigenetic modifications involved in the regulation of gene expression. The DNA methylation reaction is catalyzed by DNA methyltransferases (DNMTs). ...Recent studies have demonstrated that toxicants can affect normal development by altering DNA methylation patterns, but the mechanisms of action are poorly understood. Hence, we tested the hypothesis that developmental exposure to TCDD affects dnmt gene expression patterns. Zebrafish embryos were exposed to 5nM TCDD for 1h from 4 to 5h post-fertilization (hpf) and sampled at 12, 24, 48, 72, and 96 hpf to determine dnmt gene expression and DNA methylation patterns. We performed a detailed analysis of zebrafish dnmt gene expression during development and in adult tissues. Our results demonstrate that dnmt3b genes are highly expressed in early stages of development, and dnmt3a genes are more abundant in later stages. TCDD exposure upregulated dnmt1 and dnmt3b2 expression, whereas dnmt3a1, 3b1, and 3b4 are downregulated following exposure. We did not observe any TCDD-induced differences in global methylation or hydroxymethylation levels, but the promoter methylation of aryl hydrocarbon receptor (AHR) target genes was altered. In TCDD-exposed embryos, AHR repressor a (ahrra) and c-fos promoters were differentially methylated. To characterize the TCDD effects on DNMTs, we cloned the dnmt promoters with xenobiotic response elements and conducted AHR transactivation assays using a luciferase reporter system. Our results suggest that ahr2 can regulate dnmt3a1, dnmt3a2, and dnmt3b2 expression. Overall, we demonstrate that developmental exposure to TCDD alters dnmt expression and DNA methylation patterns.
•TCDD altered the dnmt expression in a gene and developmental time-specific manner.•TCDD hypermethylated ahrra and hypomethylated c-fos proximal promoter regions.•Functional analysis suggests that ahr2 can regulate dnmt3a1, 3a2, and 3b2 expression.•Dnmt3b genes are expressed early whereas dnmt3a are abundant later in development.
Coenzyme A (CoA)-transferases catalyze transthioesterification reactions involving acyl-CoA substrates, using an active-site carboxylate to form covalent acyl anhydride and CoA thioester adducts. ...Mechanistic studies of class I CoA-transferases suggested that acyl-CoA binding energy is used to accelerate rate-limiting acyl transfers by compressing the substrate thioester tightly against the catalytic glutamate White, H., and Jencks, W. P. (1976) J. Biol. Chem. 251, 1688–1699. The class I CoA-transferase succinyl-CoA:acetate CoA-transferase is an acetic acid resistance factor (AarC) with a role in a variant citric acid cycle in Acetobacter aceti. In an effort to identify residues involved in substrate recognition, X-ray crystal structures of a C-terminally His6-tagged form (AarCH6) were determined for several wild-type and mutant complexes, including freeze-trapped acetylglutamyl anhydride and glutamyl-CoA thioester adducts. The latter shows the acetate product bound to an auxiliary site that is required for efficient carboxylate substrate recognition. A mutant in which the catalytic glutamate was changed to an alanine crystallized in a closed complex containing dethiaacetyl-CoA, which adopts an unusual curled conformation. A model of the acetyl-CoA Michaelis complex demonstrates the compression anticipated four decades ago by Jencks and reveals that the nucleophilic glutamate is held at a near-ideal angle for attack as the thioester oxygen is forced into an oxyanion hole composed of Gly388 NH and CoA N2″. CoA is nearly immobile along its entire length during all stages of the enzyme reaction. Spatial and sequence conservation of key residues indicates that this mechanism is general among class I CoA-transferases.
Many food plants accumulate oxalate, which humans absorb but do not metabolize, leading to the formation of urinary stones. The commensal bacterium Oxalobacter formigenes consumes oxalate by ...converting it to oxalyl-CoA, which is decarboxylated by oxalyl-CoA decarboxylase (OXC). OXC and the class III CoA-transferase formyl-CoA:oxalate CoA-transferase (FCOCT) are widespread among bacteria, including many that have no apparent ability to degrade or to resist external oxalate. The EvgA acid response regulator activates transcription of the Escherichia coli yfdXWUVE operon encoding YfdW (FCOCT), YfdU (OXC), and YfdE, a class III CoA-transferase that is ~30% identical to YfdW. YfdW and YfdU are necessary and sufficient for oxalate-induced protection against a subsequent acid challenge; neither of the other genes has a known function. We report the purification, in vitro characterization, 2.1-Å crystal structure, and functional assignment of YfdE. YfdE and UctC, an orthologue from the obligate aerobe Acetobacter aceti, perform the reversible conversion of acetyl-CoA and oxalate to oxalyl-CoA and acetate. The annotation of YfdE as acetyl-CoA:oxalate CoA-transferase (ACOCT) expands the scope of metabolic pathways linked to oxalate catabolism and the oxalate-induced acid tolerance response. FCOCT and ACOCT active sites contain distinctive, conserved active site loops (the glycine-rich loop and the GNxH loop, respectively) that appear to encode substrate specificity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Early piscine life stages are sensitive to polycyclic aromatic hydrocarbon (PAH) exposure, which can cause pericardial effusion and craniofacial malformations. We previously reported that certain ...combinations of PAHs cause synergistic developmental toxicity, as observed with coexposure to the aryl hydrocarbon receptor agonist β-naphthoflavone (BNF) and cytochrome P4501A inhibitor α-naphthoflavone (ANF). Herein, we hypothesized that oxidative stress is a component of this toxicity. We examined induction of antioxidant genes in zebrafish embryos (Danio rerio) exposed to BNF or ANF individually, a BNF + ANF combination, and a prooxidant positive control, tert-butylhydroperoxide (tBOOH). We measured total glutathione (GSH) and attempted to modulate deformities using the GSH synthesis inhibitor L-buthionine (S,R)-sulfoximine (BSO) and increase GSH pools with N-acetyl cysteine (NAC). In addition, we used a morpholino to knockdown expression of the antioxidant response element transcription factor NRF2 to determine if this would alter gene expression or increase deformity severity. BNF + ANF coexposure significantly increased expressions of superoxide dismutase 1 and 2, glutathione peroxidase 1, pi class glutathione-s-transferase, and glutamate cysteine-ligase to a greater extent than tBOOH, BNF, or ANF alone. BSO pretreatment decreased some GSH levels, but did not worsen deformities, nor did NAC diminish toxicity. Knockdown of NRF2 increased mortality following tBOOH challenge, prevented significant upregulation of antioxidant genes following both tBOOH and BNF + ANF exposures, and exacerbated BNF + ANF–related deformities. Collectively, these findings demonstrate that antioxidant responses are a component of PAH synergistic developmental toxicity and that NRF2 is protective against prooxidant and PAH challenges during development.
DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases ...locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT‐like repeat (HLR) fold. AlkD uses a unique non‐base‐flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3‐methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non‐base‐flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin‐like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3‐methylcytosine (3mC) and N1‐methyladenine (1mA), which are also repaired by AlkB‐catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.
Synopsis
Contrary to most other DNA glycosylases in base excision repair, some bacterial enzymes do not flip out damaged bases for excision. The structure of AlkC reveals a new non‐base‐flipping mechanism involving DNA kinking and an immunoglobulin (Ig)‐like DNA binding domain.
Crystallization of a catalytic intermediate complex shows that AlkC does not use a base‐flipping mechanism to access alkylated nucleobases.
AlkC projects catalytic residues into a sharp DNA helix kink induced by its heat‐like repeats and Ig‐like domain.
The AlkC Ig‐like fold binds DNA differently than Ig‐like folds in eukaryotic transcription factors, and is conserved in the majority of AlkC orthologs.
Differences in the active sites and other elements of their HEAT‐like repeat domains explain the unique substrate specificities of AlkC and AlkD, a distinct non‐base‐flipping glycosylase.
DNA kinking by HEAT‐like repeats and an immunoglobulin‐like domain exemplify a new mechanism for bulky lesion exposure and excision in the absence of base‐flipping.
For qualitative researchers, selecting appropriate sites in which to conduct interviews may seem to be a relatively simple research design issue. In fact it is a complicated decision with ...wide-reaching implications. In this paper, we argue that the interview site itself embodies and constitutes multiple scales of spatial relations and meaning, which construct the power and positionality of participants in relation to the people, places, and interactions discussed in the interview. We illustrate how observation and analysis of interview sites can offer new insights with respect to research questions, help researchers understand and interpret interview material, and highlight particular ethical considerations that researchers need to address.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Abstract The increased use of silver nanoparticles in consumer and medical products has led to elevated human and environmental exposures. Silver nanoparticles act as antibacterial/antifungal agents ...by releasing Ag+ and recent studies show that Ag+ impairs neural cell replication and differentiation in culture, suggesting that in vivo exposures could compromise neurodevelopment. To determine whether Ag+ impairs development in vivo, we examined the effects of exposure on survival, morphological, and behavioral parameters in zebrafish embryos and larvae. We exposed zebrafish from 0 to 5 days post-fertilization to concentrations of Ag+ ranging from 10 nM to 100 µM in order to assess effects on survival and early embryonic development. We then tested whether concentrations below the threshold for dysmorphology altered larval behavior and subsequent survival. Ag+ concentrations ≥ 3 µM significantly reduced embryonic survival, whereas 1 µM delayed hatching with no effect on survival. Reducing the concentration to as low as 0.1 µM delayed the inflation of the swim bladder without causing gross dysmorphology or affecting hatching. At this concentration, swimming activity was impaired, an effect that persisted past the point where swim bladder inflation became normal; in contrast, general motor function was unaffected. The early behavioral impairment was then predictive of subsequent decreases in survival. Ag+ is a developmental toxicant at concentrations only slightly above allowable levels. At low concentrations, Ag+ acts as a neurobehavioral toxicant even in the absence of dysmorphology.
Job satisfaction and its antecedents and outcomes are important areas of focus in the social sciences research, and higher education is no exception. The importance of this issue has grown during the ...COVID-19 pandemic. For this reason, using a cross-national study conducted in Malaysia and Japan, we collected data on lecturers’ job satisfaction and two of its outcomes, namely, academic motivation and individual-level organizational citizenship behavior (OCBI) to test our evidence-based theoretical model, which explains the relationships between these variables. We also added age, gender, and tenure as covariates to our model. Our partial least squares structural equation modeling estimation results at the aggregate and country levels showed that the effect of job satisfaction on OCBI was mainly transmitted through academic motivation. We also observed that Malaysian and Japanese lecturers did not show a statistical difference in terms of the relationships described between the variables in our model. Additionally, the relationship between academic motivation and OCBI was nonlinear based on the data from the Malaysian sample, and we explained this phenomenon from both theoretical and practical/policy perspectives. Moreover, our results showed that age plays an important role in the model when it is estimated using data from the Malaysian higher education system. We discussed our findings in detail in terms of theoretical and practical implications.
PDF
