Search Results (29)

A restriction enzyme PsaI, an isoschizomer of the type II restriction endonuclease HindIII, has been purified to homogeneity from Gram-negative bacilli Pseudomonas anguilliseptica KM9 found in a wastewater treatment plant in Poland. Experimental data revealed that R.PsaI is highly active in the presence of Co²⁺, Mg²⁺, and Zn²⁺ and reached a maximal level of activity between 2.5 and 10 mM while its activity was significantly decreased in the presence of Ca²⁺, Fe²⁺, Mn²⁺, and Ni²⁺. Moreover, we found that the purified R.PsaI did not require NaCl for enzyme activity. Restriction cleavage analysis followed by sequencing confirmed 5′-AAGCTT-3′ as the recognition site. The genes for restriction–modification system PsaI were identified and characterized. Downstream of the psaIM gene, we noticed an ORF that shares extensive similarity with recombinase family protein specifically involved in genome rearrangements. Sequence analysis revealed that the PsaI R-M gene complex showed striking nucleotide sequence similarity (>98%) with the genes of the PanI R-M system from a P. anguilliseptica MatS1 strain identified in a soil sample from Sri Lanka. Full article

Apurinic/apyrimidinic (AP) sites are endogenous DNA lesions widespread in human cells. Having no nucleobases, they are noncoding and promutagenic. AP site repair is generally initiated through strand incision by AP endonuclease 1 (APE1). Although AP sites’ repair in regular B-DNA has been studied extensively, their processing in G-quadruplexes (G4s) has received much less attention. Here, we used the hTERT promoter region that is capable of forming three stacked parallel G4s to understand how AP sites can influence higher-order quadruplex folding and stability and how a G4 affects the efficiency of human APE1-mediated AP site processing. We designed a series of synthetic single- and double-stranded DNA constructs of varying lengths containing a stable AP site analog in both G- and C-rich strands at positions corresponding to somatic driver mutations. Using circular dichroism, we studied the effect of the AP site on hTERT G4 structure and stability. Bio-layer interferometry and gel-based approaches were employed to characterize APE1 binding to the designed DNA substrates and AP site processing. It was shown that (i) an AP site leads to G4 destabilization, which depends on the lesion location in the G4 scaffold; (ii) APE1 binds tightly to hTERT G4 structure but exhibits greatly reduced cleavage activity at AP sites embedded in the quadruplex; and (iii) a clear correlation was revealed between AP site-induced hTERT G4 destabilization and APE1 activity. We can hypothesize that reduced repair of AP sites in the hTERT G4 is one of the reasons for the high mutation rate in this promoter region. Full article

Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1′s regulatory functions extend to many cellular processes including the regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins—PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase β, DNA ligase III, and XRCC1—did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either. Full article

The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPy^TM (“SAR by X-ray Poses Quickly”) platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or “undruggable” targets, allows for (i) hit generation; (ii) the mapping of protein–ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target. Full article

Restriction endonucleases are a component of restriction–modification systems, where the main biological function is to protect bacterial cells from incoming foreign DNA molecules. There are four main types of restriction enzymes (types I, II, III, and IV), which differ in protein composition, cofactor requirements, and mode of action. The most studied are representatives of type II, which specifically recognize DNA sequences of 4–8 bp and catalyze DNA cleavage within these sequences or not far from them. The exceptional precision of type II enzymes has made them indispensable for DNA manipulations. Although hundreds of DNA restriction enzymes are currently known, there is still a need for enzymes that recognize new DNA targets. For this reason, the discovery of new natural restriction endonucleases and rational design of their properties (to obtain enzymes with high specificity for a unique nucleotide sequence at a restriction site and without nonspecific activity) will expand the list of enzymes for use in biotechnology and genetic engineering. This review briefly touches upon the main types of restriction endonucleases, their classification, nomenclature, and typical properties, and it concisely describes approaches to the construction of enzymes with altered properties. Full article

Baloxavir marboxil is a creative antiviral drug for influenza A and B viruses with a novel mechanism of action. In this study, three series comprising a total of 21 previously unreported target compounds were designed and synthesized. The drug-likeness of these compounds was evaluated by molecular docking, PAINS-Remover and SwissADME. The inhibitory effect and affinity of the compounds on the cap-dependent endonuclease activity of the influenza virus were evaluated. Compounds I-4, II-1~II-9 and compound III-8 showed similar inhibitory activity to baloxavir (7.45 μM) on the cap-dependent endonuclease. In particular, compounds I-4 (3.29 μM) and II-2 (1.46 μM) showed strong cap-dependent endonuclease inhibitory activity. The structure–activity relationship studies showed that the inhibitive effect of the compounds on endonuclease was enhanced when the dibenzothiepin rings were substituted by diphenylmethyl containing chiral-center electron-withdrawing groups, dibenzocycloheptane, dihydrodibenzo[b,e]oxepin, and five-member heterocycles containing aryl substitution. Full article

Alternaria black spot disease on pecan is caused by the opportunistic pathogen Alternaria alternata and poses a serious threat to the local South African and global pecan industry. Several diagnostic molecular marker applications have been established and used in the screening of various fungal diseases worldwide. The present study investigated the potential for polymorphism within samples of A. alternata isolates obtained from eight different geographical locations in South Africa. Pecan (Carya illinoinensis) leaves, shoots, and nuts-in-shuck with Alternaria black spot disease were sampled, and 222 A. alternata isolates were retrieved. For rapid screening to identify Alternaria black spot pathogens, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis of the Alternaria major allergen (Alt a1) gene region was used, followed by the digestion of the amplicons with HaeIII and HinfI endonucleases. The assay resulted in five (HaeIII) and two (HinfI) band patterns. Unique banding patterns from the two endonucleases showed the best profile and isolates were grouped into six clusters using a UPGMA (unweighted pair group method with arithmetic averages) distance matrix (Euclidean) dendrogram method on R-Studio. The analysis confirmed that the genetic diversity of A. alternata does not depend on host tissues or the pecan cultivation region. The grouping of selected isolates was confirmed by DNA sequence analysis. The Alt a1 phylogeny corroborated no speciation within the dendrogram groups and showed 98–100% bootstrap similarity. This study reports the first documented rapid and reliable technique for routine screening identification of pathogens causing Alternaria black spot in South Africa. Full article

The human pathogen Neisseria gonorrhoeae uses a homologous recombination to undergo antigenic variation and avoid an immune response. The surface protein pilin (PilE) is one of the targets for antigenic variation that can be regulated by N. gonorrhoeae mismatch repair (MMR) and a G-quadruplex (G4) located upstream of the pilE promoter. Using bioinformatics tools, we found a correlation between pilE variability and deletion of DNA regions encoding ngMutS or ngMutL proteins, the main participants in N. gonorrhoeae methyl-independent MMR. To understand whether the G4 structure could affect the ngMutL-mediated regulation of pilin antigenic variation, we designed several synthetic pilE G4-containing oligonucleotides, differing in length, and related DNA duplexes. Using CD measurements and biochemical approaches, we have showed that (i) ngMutL preferentially binds to pilE G4 compared to DNA duplex, although the latter is a cognate substrate for ngMutL endonuclease, (ii) protein binding affinity decreases with shortening of quadruplex-containing and duplex ligands, (iii) the G4 structure inhibits ngMutL-induced DNA nicking and modulates cleavage positions; the enzyme does not cleave DNA within G4, but is able to bypass this noncanonical structure. Thus, pilE G4 may regulate the efficiency of pilin antigenic variation by quadruplex binding to ngMutL and suppression of homologous recombination. Full article

Homologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In Escherichia coli, DSB repair is initiated by the RecBCD enzyme that resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand (ss) DNA tails. SSG repair is mediated by the RecFOR protein complex that loads RecA onto the ssDNA segment of gaped duplex. In both repair pathways, RecA catalyses reactions of homologous DNA pairing and strand exchange, while RuvABC complex and RecG helicase process recombination intermediates. In this work, we have characterised cytological changes in various recombination mutants of E. coli after three different DNA-damaging treatments: (i) expression of I-SceI endonuclease, (ii) γ-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the ruvABC, recG, and ruvABC recG mutants. After I-SceI expression and γ-irradiation, this phenotype was efficiently suppressed by the recB mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the recB mutation abolished cytological defects of recG mutants and also partially suppressed the cytological defects of ruvABC recG mutants. However, neither recB nor recO mutation alone could suppress the cytological defects of UV-irradiated ruvABC mutants. The suppression was achieved only by simultaneous inactivation of the recB and recO genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated ruvABC mutants largely result from defective processing of stalled replication forks. The results of this study show that chromosome morphology is a valuable marker in genetic analyses of recombinational repair in E. coli. Full article

The occlusion bodies (OBs) of lepidopteran nucleopolyhedroviruses can persist in soil for extended periods before being transported back on to the foliage for transmission to the host insect. A sensitive insect bioassay technique was used to detect OBs of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) in 186 soil samples collected from maize fields in the southern Mexican states of Chiapas, Tabasco, Campeche, Yucatán, and Quintana Roo, as well Belize and Guatemala. Overall, 35 (18.8%) samples proved positive for SfMNPV OBs. The frequency of OB-positive samples varied significantly among Mexican states and countries (p < 0.05). Between 1.7 and 4.4% of S. frugiperda larvae that consumed OB-positive samples died from polyhedrosis disease. Restriction endonuclease analysis using PstI and HindIII confirmed that the soil-derived isolates were strains of SfMNPV and that genetic diversity was evident among the isolates. The prevalence of OB-positive soil samples did not differ with altitude or extension (area) of the maize field, but it was significantly higher in fields with the presence of living maize plants compared to those containing dead plants or crop residues (p < 0.05). Georeferenced soil samples were used to identify soil types on digitized soil maps. Lithosol and Luvisol soils had a higher than average prevalence of OB-positive samples (42–45% positive) (p = 0.006), as did Andosol, Gleysol, and Vertisol soils (33–60% OB-positive), although the sample sizes were small (<5 samples) for the latter three soils. In contrast, Cambisol soils had a lower than average prevalence of OB-positive samples (5% positive). Bioassays on Acrisol, Fluvisol, Phaeozem, and Rendzina soils resulted in intermediate levels of OB-positive samples. We conclude that certain soil types may favor OB persistence and virus-mediated biological pest control. The soil is also likely to provide a valuable source of genetic diversity for the design of virus-based insecticides against this pest. Full article

Oxidative DNA base lesions in DNA are repaired through the base excision repair (BER) pathway, which consequently plays a vital role in the maintenance of genome integrity and in suppressing mutagenesis. 8-oxoguanine DNA glycosylase (OGG1), endonuclease III-like protein 1 (NTH1), and the endonuclease VIII-like proteins 1–3 (NEIL1–3) are the key enzymes that initiate repair through the excision of the oxidized base. We have previously identified that the E3 ubiquitin ligase tripartite motif 26 (TRIM26) controls the cellular response to oxidative stress through regulating both NEIL1 and NTH1, although its potential, broader role in BER is unclear. We now show that TRIM26 is a central player in determining the response to different forms of oxidative stress. Using siRNA-mediated knockdowns, we demonstrate that the resistance of cells to X-ray radiation and hydrogen peroxide generated as a consequence of trim26 depletion can be reversed through suppression of selective DNA glycosylases. In particular, a knockdown of neil1 or ogg1 can enhance sensitivity and DNA repair rates in response to X-rays, whereas a knockdown of neil1 or neil3 can produce the same effect in response to hydrogen peroxide. Our study, therefore, highlights the importance of TRIM26 in balancing cellular DNA glycosylase levels required for an efficient BER response. Full article

Radish is a typical self-incompatible crop. The rapid and accurate identification of S haplotypes can circumvent the blindness of the hybrid combination process, which is critical in radish heterosis utilization and the breeding of new varieties. In this study, based on the gene sequence which encodes the S-locus receptor kinase (SRK) of radish, and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, the S haplotypes were identified among 79 cultivated radish genotypes. The PCR results indicated that 79 radish genotypes could be divided into 48 Class I, 13 Class II, and 17 Class I/II S haplotypes. Sequence alignment confirmed that the Class I materials contained 19 S haplotypes, of which three haplotypes (‘NAU-S53’, ‘NAU-S54’ and ‘NAU-S55’) were identified for the first time in radish. After digestion using the Hinf I restriction endonuclease, the SRK domain of DNA fragments of different genotypes showed high polymorphism. Homozygous materials S haplotypes could be quickly distinguished by the differences in the digested bands. Molecular identification of the S haplotype was highly consistent with the field pollination and pollen tube germination results. These results would provide an important approach for the rapid identification of radish S haplotypes and the efficient utilization of self-incompatibility in heterosis breeding. Full article

Endonuclease III (EndoIII) is a bifunctional DNA glycosylase with specificity for a broad range of oxidized DNA lesions. The genome of an extremely radiation- and desiccation-resistant bacterium, Deinococcus radiodurans, possesses three genes encoding for EndoIII-like enzymes (DrEndoIII1, DrEndoIII2 and DrEndoIII3), which reveal different types of catalytic activities. DrEndoIII2 acts as the main EndoIII in this organism, while DrEndoIII1 and 3 demonstrate unusual and no EndoIII activity, respectively. In order to understand the role of DrEndoIII1 and DrEndoIII3 in D. radiodurans, we have generated mutants which target non-conserved residues in positions considered essential for classic EndoIII activity. In parallel, we have substituted residues coordinating the iron atoms in the [4Fe-4S] cluster in DrEndoIII2, aiming at elucidating the role of the cluster in these enzymes. Our results demonstrate that the amino acid substitutions in DrEndoIII1 reduce the enzyme activity without altering the overall structure, revealing that the residues found in the wild-type enzyme are essential for its unusual activity. The attempt to generate catalytic activity of DrEndoIII3 by re-designing its catalytic pocket was unsuccessful. A mutation of the iron-coordinating cysteine 199 in DrEndoIII2 appears to compromise the structural integrity and induce the formation of a [3Fe-4S] cluster, but apparently without affecting the activity. Taken together, we provide important structural and mechanistic insights into the three EndoIIIs, which will help us disentangle the open questions related to their presence in D. radiodurans and their particularities. Full article

Cisplatin can induce peripheral neuropathy, which is a common complication of anti-cancer treatment and negatively impacts cancer survivors during and after completion of treatment; therefore, the mechanisms by which cisplatin alters sensory neuronal function to elicit neuropathy are the subject of much investigation. Our previous work suggests that the DNA repair activity of APE1/Ref-1, the rate-limiting enzyme of the base excision repair (BER) pathway, is critical for neuroprotection against cisplatin. A specific role for 8-oxoguanine DNA glycosylase-1 (OGG1), the glycosylase that removes the most common oxidative DNA lesion, and putative coordination of OGG1 with APE1/Ref-1 in sensory neurons, has not been investigated. We investigated whether inhibiting OGG1 glycosylase activity with the small molecule inhibitor, TH5487, and/or APE1/Ref-1 endonuclease activity with APE Repair Inhibitor III would alter the neurotoxic effects of cisplatin in sensory neuronal cultures. Sensory neuron function was assessed by calcitonin gene-related peptide (CGRP) release, as a marker of sensitivity and by neurite outgrowth. Cisplatin altered neuropeptide release in an inverse U-shaped fashion, with low concentrations enhancing and higher concentrations diminishing CGRP release. Pretreatment with BER inhibitors exacerbated the functional effects of cisplatin and enhanced 8oxo-dG and adduct lesions in the presence of cisplatin. Our studies demonstrate that inhibition of OGG1 and APE1 endonuclease activity enhances oxidative DNA damage and exacerbates neurotoxicity, thus limiting oxidative DNA damage in sensory neurons that might alleviate cisplatin-induced neuropathy. Full article

DNA methylomes of Helicobacter pylori strains are complex due to the large number of DNA methyltransferases (MTases) they possess. H. pylori J99 M.Hpy99III is a 5-methylcytosine (^m5C) MTase that converts GCGC motifs to G^m5CGC. Homologs of M.Hpy99III are found in essentially all H. pylori strains. Most of these homologs are orphan MTases that lack a cognate restriction endonuclease, and their retention in H. pylori strains suggest they have roles in gene regulation. To address this hypothesis, green fluorescent protein (GFP) reporter genes were constructed with six putative promoters that had a GCGC motif in the extended −10 region, and the expression of the reporter genes was compared in wild-type H. pylori G27 and a mutant lacking the M.Hpy99III homolog (M.HpyGIII). The expression of three of the GFP reporter genes was decreased significantly in the mutant lacking M.HpyGIII. In addition, the growth rate of the H. pylori G27 mutant lacking M.HpyGIII was reduced markedly compared to that of the wild type. These findings suggest that the methylation of the GCGC motif in many H. pylori GCGC-containing promoters is required for the robust expression of genes controlled by these promoters, which may account for the universal retention of M.Hpy99III homologs in H. pylori strains. Full article