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Fahmi, Tariq, Xiaoying Wang, Dmitry D. Zhdanov, Intisar Islam, Eugene O. Apostolov, Alena V. Savenka et Alexei G. Basnakian. « DNase I Induces Other Endonucleases in Kidney Tubular Epithelial Cells by Its DNA-Degrading Activity ». International Journal of Molecular Sciences 21, no 22 (17 novembre 2020) : 8665. http://dx.doi.org/10.3390/ijms21228665.
Texte intégralRésumé :
Endonuclease-mediated DNA fragmentation is both an immediate cause and a result of apoptosis and of all other types of irreversible cell death after injury. It is produced by nine enzymes including DNase I, DNase 2, their homologs, caspase-activated DNase (CAD) and endonuclease G (EndoG). The endonucleases act simultaneously during cell death; however, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most abundant of endonucleases, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK-52E cells were transfected with the DNase I gene or its inactive mutant in a pECFP expression vector, while control cells were transfected with the empty vector. mRNA expression of all nine endonucleases was studied using real-time RT-PCR; DNA strand breaks in endonuclease genes were determined by PCR and protein expression of the enzymes was measured by Western blotting and quantitative immunocytochemistry. Our data showed that DNase I, but not its inactive mutant, induces all other endonucleases at varying time periods after transfection, causes DNA breaks in endonuclease genes, and elevates protein expression of several endonucleases. This is the first evidence that endonucleases seem to be induced by the DNA-degrading activity of DNase I.
2
Kamisugi, Y., Y. Ikeda, M. Ohno, M. Minezawa et K. Fukui. « In situ digestion of barley chromosomes with restriction endonucleases ». Genome 35, no 5 (1 octobre 1992) : 793–98. http://dx.doi.org/10.1139/g92-121.
Texte intégralRésumé :
In situ digestion of barley chromosomes with restriction endonucleases was examined. All the treatments with five restriction endonucleases, MboII, RsaI, HaeIII, HinfI, and DraII, showed various band patterns on the barley chromosomes. Differences were observed in the band patterns produced with different restriction endonucleases. Uneven staining patterns, similar to the band patterns by the endonuclease treatments, also appeared when the chromosomes were treated with the buffer solution without the enzyme. The band patterns observed both with and without the endonucleases were classified into the four types and the frequency of each type among the different treatments was investigated. The change of the band types along with treatment time was accelerated by the addition of the restriction endonuclease. As a result, it was concluded that there existed chromosome band patterns that were specific to the endonuclease treatments and that the buffer solution also affected to the production of the bands on the chromosomes.Key words: Hordeum vulgare L., chromosome band pattern, in situ digestion, restriction endonuclease, restriction banding.
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Petersen, Kamilla Vandsø, Cinzia Tesauro, Marianne Smedegaard Hede, Camilla Pages, Lærke Bay Marcussen, Josephine Geertsen Keller, Magnus Bugge et al. « Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts ». Sensors 22, no 20 (13 octobre 2022) : 7763. http://dx.doi.org/10.3390/s22207763.
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Restriction endonucleases are expressed in all bacteria investigated so far and play an essential role for the bacterial defense against viral infections. Besides their important biological role, restriction endonucleases are of great use for different biotechnological purposes and are indispensable for many cloning and sequencing procedures. Methods for specific detection of restriction endonuclease activities can therefore find broad use for many purposes. In the current study, we demonstrate proof-of-concept for a new principle for the detection of restriction endonuclease activities. The method is based on rolling circle amplification of circular DNA products that can only be formed upon restriction digestion of specially designed DNA substrates. By combining the activity of the target restriction endonuclease with the highly specific Cre recombinase to generate DNA circles, we demonstrate specific detection of selected restriction endonuclease activities even in crude cell extracts. This is, to our knowledge, the first example of a sensor system that allows activity measurements of restriction endonucleases in crude samples. The presented sensor system may prove valuable for future characterization of bacteria species or strains based on their expression of restriction endonucleases as well as for quantification of restriction endonuclease activities directly in extracts from recombinant cells.
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Shammas, Masood A., Hemant Koley, Sima Shah, Ramesh B. Batchu, Pierfrancesco Tassone, Kenneth C. Anderson et Nikhil C. Munshi. « Dysregulated Apurinic/Apyrimidinic Endonucleases (Ape1 and Ape2) Lead to Genetic Instability in Multiple Myeloma. » Blood 104, no 11 (16 novembre 2004) : 1418. http://dx.doi.org/10.1182/blood.v104.11.1418.1418.
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Abstract Multiple myeloma (MM) is associated with significant genomic instability. Homologous recombination (HR), which is elevated in MM, is considered to be responsible for this instability. As endonucleases play an important role in mediating HR, here we have evaluated the role of endonuclease in biology and progression of MM. Gene expression profile using Affymetrix U133 array showed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Immunocytochemistry confirmed upregulation of Ape1 protein in MM cell lines. A Plasmid degradation assay confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease activity, the degradation assay was carried out in the presence of specific endonuclease inhibitors. Harmane and methoxyamine (MA), specific inhibitors of apurinic/apyrimidinic endonucleases effectively inhibited significant endonuclease activity, while other endonuclease inhibitors ACPD and FK506 had minimal effects, confirming predominant role of apurinic/apyrimidinic endonucleases (APE) in mediating increased endonuclease activity in MM. We investigated the role of elevated APE endonuclease activity on DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. Inhibition of endonuclease by methoxyamine suppressed HR activity by 85 ± 2% in MM cells. Next, we evaluated whether inhibition of HR by methoxyamine can affect the frequency of acquisition of new genetic changes in MM cells using single nucleotide polymorphism (SNP) arrays (Affymetrix) as indicator of genomic instability. In three independent experiments, methoxyamine reduced the acquisition of new loss of heterozygocity (LOH) loci by an average of 71%. These data suggest that the dysregulated APE endonucleases contribute significantly to the genomic instability, acquisition of new mutations and progression of MM and provides the rationale for targeting endonuclease activity to prevent disease progression including development of drug resistance.
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Landthaler, Markus, Nelson C. Lau et David A. Shub. « Group I Intron Homing in Bacillus Phages SPO1 and SP82 : a Gene Conversion Event Initiated by a Nicking Homing Endonuclease ». Journal of Bacteriology 186, no 13 (1 juillet 2004) : 4307–14. http://dx.doi.org/10.1128/jb.186.13.4307-4314.2004.
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ABSTRACT Many group I introns encode endonucleases that promote intron homing by initiating a double-stranded break-mediated homologous recombination event. In this work we describe intron homing in Bacillus subtilis phages SPO1 and SP82. The introns encode the DNA endonucleases I-HmuI and I-HmuII, respectively, which belong to the H-N-H endonuclease family and possess nicking activity in vitro. Coinfections of B. subtilis with intron-minus and intron-plus phages indicate that I-HmuI and I-HmuII are required for homing of the SPO1 and SP82 introns, respectively. The homing process is a gene conversion event that does not require the major B. subtilis recombination pathways, suggesting that the necessary functions are provided by phage-encoded factors. Our results provide the first examples of H-N-H endonuclease-mediated intron homing and the first demonstration of intron homing initiated by a nicking endonuclease.
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GRISHIN, ALEXANDER, INES FONFARA, ANDREI ALEXEEVSKI, SERGEI SPIRIN, OLGA ZANEGINA, ANNA KARYAGINA, DANIIL ALEXEYEVSKY et WOLFGANG WENDE. « IDENTIFICATION OF CONSERVED FEATURES OF LAGLIDADG HOMING ENDONUCLEASES ». Journal of Bioinformatics and Computational Biology 08, no 03 (juin 2010) : 453–69. http://dx.doi.org/10.1142/s0219720010004665.
Texte intégralRésumé :
LAGLIDADG family of homing endonucleases are rare-cutting enzymes which recognize long target sequences and are of great interest in genome engineering. Despite advances in homing endonuclease engineering, effective methods of broadening the range of cleaved sequences are still lacking. Here, we present a study of conserved structural features of LAGLIDADG homing endonucleases that might aid further development of such methods. The protein–DNA interface of LAGLIDADG homing endonucleases differs considerably with the particular nuclease, and the analysis of conserved protein–DNA interactions could not identify any residues crucial for DNA binding and common to most nucleases of the family. For the homing endonuclease PI-SceI, a comparison of structural and experimental data derived from literature helped to identify 23 residues that are likely to be important for DNA binding. Analysis of the LAGLIDADG domain dimerization interface allowed the choosing of six positions that contribute to dimerization specificity most, while comparison of 446 sequences of LAGLIDADG endonucleases revealed groups of residues in these positions that appear to be most favorable for dimerization.
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Everett, Blake A., Lauren A. Litzau, Kassidy Tompkins, Ke Shi, Andrew Nelson, Hideki Aihara, Robert L. Evans et Wendy R. Gordon. « Crystal structure of the Wheat dwarf virus Rep domain ». Acta Crystallographica Section F Structural Biology Communications 75, no 12 (27 novembre 2019) : 744–49. http://dx.doi.org/10.1107/s2053230x19015796.
Texte intégralRésumé :
The Rep domain of Wheat dwarf virus (WDV Rep) is an HUH endonuclease involved in rolling-circle replication. HUH endonucleases coordinate a metal ion to enable the nicking of a specific ssDNA sequence and the subsequent formation of an intermediate phosphotyrosine bond. This covalent protein–ssDNA adduct makes HUH endonucleases attractive fusion tags (HUH-tags) in a diverse number of biotechnological applications. Solving the structure of an HUH endonuclease in complex with ssDNA will provide critical information about ssDNA recognition and sequence specificity, thus enabling rationally engineered protein–DNA interactions that are programmable. The structure of the WDV Rep domain reported here was solved in the apo state from a crystal diffracting to 1.24 Å resolution and represents an initial step in the direction of solving the structure of a protein–ssDNA complex.
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Bultmann, H., et R. Mezzanotte. « Characterization and origin of extrachromosomal DNA granules in Sarcophaga bullata ». Journal of Cell Science 88, no 3 (1 octobre 1987) : 327–34. http://dx.doi.org/10.1242/jcs.88.3.327.
Texte intégralRésumé :
We have used endonuclease treatment in situ, followed by Giemsa or ethidium bromide staining, for mapping repetitive sequences on the chromosomes of the flesh fly Sarcophaga bullata and thus for studying extrachromosomal DNA granules in this species. All three restriction enzymes employed (HaeIII, A1uI and HindIII) show the same cytological effects, except for a single interstitial band. In both polytene and mitotic chromosomes, chromatin resistant to these endonucleases presumably includes at least three endonucleases presumably includes at least three previously unrecognized buoyant density satellites (1.663, 1.670 and 1.692 g ml-1 in neutral CsCl), and is predominantly localized in the pericentric regions of all five autosomes. Mitotic treated chromosomes show that the entire rod-shaped X chromosome, but no part of the dot-like Y chromosome, consists of endonuclease-resistant chromatin. The most unusual heterochromatic component of polytene nuclei in this species, the ‘extrachromosomal DNA granules’, are also entirely resistant to digestion with endonucleases. We think that these DNA granules represent dispersed X chromatin and not, as previously assumed, extruded autosomal heterochromatin.
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Jordano-Raya, Marina, Cristina Beltrán-Melero, M. Dolores Moreno-Recio, M. Isabel Martínez-Macías, Rafael R. Ariza, Teresa Roldán-Arjona et Dolores Córdoba-Cañero. « Complementary Functions of Plant AP Endonucleases and AP Lyases during DNA Repair of Abasic Sites Arising from C:G Base Pairs ». International Journal of Molecular Sciences 22, no 16 (16 août 2021) : 8763. http://dx.doi.org/10.3390/ijms22168763.
Texte intégralRésumé :
Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively.
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Carnes, Jason, Carmen Zelaya Soares, Carey Wickham et Kenneth Stuart. « Endonuclease Associations with Three Distinct Editosomes in Trypanosoma brucei ». Journal of Biological Chemistry 286, no 22 (7 avril 2011) : 19320–30. http://dx.doi.org/10.1074/jbc.m111.228965.
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Three distinct editosomes, typified by mutually exclusive KREN1, KREN2, or KREN3 endonucleases, are essential for mitochondrial RNA editing in Trypanosoma brucei. The three editosomes differ in substrate endoribonucleolytic cleavage specificity, which may reflect the vast number of editing sites that need insertion or deletion of uridine nucleotides (Us). Each editosome requires the single RNase III domain in each endonuclease for catalysis. Studies reported here show that the editing endonucleases do not form homodimeric domains, and may therefore function as intermolecular heterodimers, perhaps with KREPB4 and/or KREPB5. Editosomes isolated via TAP tag fused to KREPB6, KREPB7, or KREPB8 have a common set of 12 proteins. In addition, KREN3 is only found in KREPB6 editosomes, KREN2 is only found in KREPB7 editosomes, and KREN1 is only found in KREPB8 editosomes. These are the same associations previously found in editosomes isolated via the TAP-tagged endonucleases KREN1, KREN2, or KREN3. Furthermore, TAP-tagged KREPB6, KREPB7, and KREPB8 complexes isolated from cells in which expression of their respective endonuclease were knocked down were disrupted and lacked the heterotrimeric insertion subcomplex (KRET2, KREPA1, and KREL2). These results and published data suggest that KREPB6, KREPB7, and KREPB8 associate with the deletion subcomplex, whereas the KREN1, KREN2, and KREN3 endonucleases associate with the insertion subcomplex.
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Daley, James M., Chadi Zakaria et Dindial Ramotar. « The endonuclease IV family of apurinic/apyrimidinic endonucleases ». Mutation Research/Reviews in Mutation Research 705, no 3 (décembre 2010) : 217–27. http://dx.doi.org/10.1016/j.mrrev.2010.07.003.
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Anzai, N., H. Kawabata, T. Hirama, H. Masutani, Y. Ueda, Y. Yoshida et M. Okuma. « Types of nuclear endonuclease activity capable of inducing internucleosomal DNA fragmentation are completely different between human CD34+ cells and their granulocytic descendants ». Blood 86, no 3 (1 août 1995) : 917–23. http://dx.doi.org/10.1182/blood.v86.3.917.917.
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Abstract A hallmark of apoptosis is internucleosomal DNA fragmentation resulting from the activation of endonucleases. We characterized the endonuclease activity of human myeloid cell nuclei that cleaved their own nuclear chromatin to oligonucleosomal length fragments. Polymorphonuclear leukocytes (PMNs) of normal peripheral blood contained both Ca2+/Mg(2+)- dependent and DNase II-like acidic endonuclease activities in their nuclei. Immature myeloid cells of normal bone marrow at various stages of granulocytic maturation had similar nuclease activities. In contrast, a clear difference was shown in the circulating CD34+ cells, in that only Mg(2+)-dependent, Ca(2+)-independent endonuclease activity was detected. Consistent with these findings is the emergence of the Ca2+/Mg(2+)-dependent and acidic endonuclease concomitantly with the disappearance of the Mg(2+)-dependent endonuclease when CD34+ cells were induced to differentiate in vitro toward granulocytes. Leukemic cell lines of all lineages also had Mg(2+)-dependent nuclease activity. Our results suggest an association of the Mg(2+)-dependent endonuclease with hematopoietic progenitor cells and that the relative activities of the nuclear nuclease in human myeloid cells change substantially during granulocytic differentiation.
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Anzai, N., H. Kawabata, T. Hirama, H. Masutani, Y. Ueda, Y. Yoshida et M. Okuma. « Types of nuclear endonuclease activity capable of inducing internucleosomal DNA fragmentation are completely different between human CD34+ cells and their granulocytic descendants ». Blood 86, no 3 (1 août 1995) : 917–23. http://dx.doi.org/10.1182/blood.v86.3.917.bloodjournal863917.
Texte intégralRésumé :
A hallmark of apoptosis is internucleosomal DNA fragmentation resulting from the activation of endonucleases. We characterized the endonuclease activity of human myeloid cell nuclei that cleaved their own nuclear chromatin to oligonucleosomal length fragments. Polymorphonuclear leukocytes (PMNs) of normal peripheral blood contained both Ca2+/Mg(2+)- dependent and DNase II-like acidic endonuclease activities in their nuclei. Immature myeloid cells of normal bone marrow at various stages of granulocytic maturation had similar nuclease activities. In contrast, a clear difference was shown in the circulating CD34+ cells, in that only Mg(2+)-dependent, Ca(2+)-independent endonuclease activity was detected. Consistent with these findings is the emergence of the Ca2+/Mg(2+)-dependent and acidic endonuclease concomitantly with the disappearance of the Mg(2+)-dependent endonuclease when CD34+ cells were induced to differentiate in vitro toward granulocytes. Leukemic cell lines of all lineages also had Mg(2+)-dependent nuclease activity. Our results suggest an association of the Mg(2+)-dependent endonuclease with hematopoietic progenitor cells and that the relative activities of the nuclear nuclease in human myeloid cells change substantially during granulocytic differentiation.
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Karvelis, Tautvydas, Giedrius Gasiunas et Virginijus Siksnys. « Programmable DNA cleavage in vitro by Cas9 ». Biochemical Society Transactions 41, no 6 (20 novembre 2013) : 1401–6. http://dx.doi.org/10.1042/bst20130164.
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The ternary Cas9–crRNA–tracrRNA complex (Cas9t) of the Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) system functions as an Mg2+-dependent RNA-directed DNA endonuclease that locates its DNA target guided by the crRNA (CRISPR RNA) in the tracrRNA–crRNA structure and introduces a double-strand break at a specific site in DNA. The simple modular organization of Cas9t, where specificity for the DNA target is encoded by a small crRNA and the cleavage reaction is executed by the Cas9 endonuclease, provides a versatile platform for the engineering of universal RNA-directed DNA endonucleases. By altering the crRNA sequence within the Cas9t complex, programmable endonucleases can be designed for both in vitro and in vivo applications. Cas9t has been recently employed as a gene-editing tool in various eukaryotic cell types. Using Streptococcus thermophilus Cas9t as a model system, we demonstrate the feasibility of Cas9t as a programmable molecular tool for in vitro DNA manipulations.
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Takai, Ken, et Koki Horikoshi. « Molecular Phylogenetic Analysis of Archaeal Intron-Containing Genes Coding for rRNA Obtained from a Deep-Subsurface Geothermal Water Pool ». Applied and Environmental Microbiology 65, no 12 (1 décembre 1999) : 5586–89. http://dx.doi.org/10.1128/aem.65.12.5586-5589.1999.
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ABSTRACT Molecular phylogenetic analysis of a naturally occurring microbial community in a deep-subsurface geothermal environment indicated that the phylogenetic diversity of the microbial population in the environment was extremely limited and that only hyperthermophilic archaeal members closely related to Pyrobaculum were present. All archaeal ribosomal DNA sequences contained intron-like sequences, some of which had open reading frames with repeated homing-endonuclease motifs. The sequence similarity analysis and the phylogenetic analysis of these homing endonucleases suggested the possible phylogenetic relationship among archaeal rRNA-encoded homing endonucleases.
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Yang, Zhi-Hui, Ji-Xiang Huang et Yi-Jian Yao. « Autoscreening of Restriction Endonucleases for PCR-Restriction Fragment Length Polymorphism Identification of Fungal Species, with Pleurotus spp. as an Example ». Applied and Environmental Microbiology 73, no 24 (26 octobre 2007) : 7947–58. http://dx.doi.org/10.1128/aem.00842-07.
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ABSTRACT A molecular method based on PCR-restriction fragment length polymorphism (RFLP) analysis of internal transcribed spacer (ITS) ribosomal DNA sequences was designed to rapidly identify fungal species, with members of the genus Pleurotus as an example. Based on the results of phylogenetic analysis of ITS sequences from Pleurotus, a PCR-RFLP endonuclease autoscreening (PRE Auto) program was developed to screen restriction endonucleases for discriminating multiple sequences from different species. The PRE Auto program analyzes the endonuclease recognition sites and calculates the sizes of the fragments in the sequences that are imported into the program in groups according to species recognition. Every restriction endonuclease is scored through the calculation of the average coefficient for the sequence groups and the average coefficient for the sequences within a group, and then virtual electrophoresis maps for the selected restriction enzymes, based on the results of the scoring system, are displayed for the rapid determination of the candidate endonucleases. A total of 85 haplotypes representing 151 ITS sequences were used for the analysis, and 2,992 restriction endonucleases were screened to find the candidates for the identification of species. This method was verified by an experiment with 28 samples representing 12 species of Pleurotus. The results of the digestion by the restriction enzymes showed the same patterns of DNA fragments anticipated by the PRE Auto program, apart from those for four misidentified samples. ITS sequences from 14 samples (of which nine sequences were obtained in this study), including four originally misidentified samples, confirmed the species identities revealed by the PCR-RFLP analysis. The method developed here can be used for the identification of species of other living microorganisms.
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Davletgildeeva, Anastasiia T., Alexandra A. Kuznetsova, Darya S. Novopashina, Alexander A. Ishchenko, Murat Saparbaev, Olga S. Fedorova et Nikita A. Kuznetsov. « Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes ». International Journal of Molecular Sciences 23, no 5 (6 mars 2022) : 2869. http://dx.doi.org/10.3390/ijms23052869.
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Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5′ to an AP-site; can recognize and process some damaged nucleosides; and possess 3′-phosphodiesterase, 3′-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions’ concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3′-5′ exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes.
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Jones, Rhian, Sana Lessoued, Kristina Meier, Stéphanie Devignot, Sergio Barata-García, Maria Mate, Gabriel Bragagnolo, Friedemann Weber, Maria Rosenthal et Juan Reguera. « Structure and function of the Toscana virus cap-snatching endonuclease ». Nucleic Acids Research 47, no 20 (4 octobre 2019) : 10914–30. http://dx.doi.org/10.1093/nar/gkz838.
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Abstract Toscana virus (TOSV) is an arthropod-borne human pathogen responsible for seasonal outbreaks of fever and meningoencephalitis in the Mediterranean basin. TOSV is a segmented negative-strand RNA virus (sNSV) that belongs to the genus phlebovirus (family Phenuiviridae, order Bunyavirales), encompassing other important human pathogens such as Rift Valley fever virus (RVFV). Here, we carried out a structural and functional characterization of the TOSV cap-snatching endonuclease, an N terminal domain of the viral polymerase (L protein) that provides capped 3′OH primers for transcription. We report TOSV endonuclease crystal structures in the apo form, in complex with a di-ketoacid inhibitor (DPBA) and in an intermediate state of inhibitor release, showing details on substrate binding and active site dynamics. The structure reveals substantial folding rearrangements absent in previously reported cap-snatching endonucleases. These include the relocation of the N terminus and the appearance of new structural motifs important for transcription and replication. The enzyme shows high activity rates comparable to other His+ cap-snatching endonucleases. Moreover, the activity is dependent on conserved residues involved in metal ion and substrate binding. Altogether, these results bring new light on the structure and function of cap-snatching endonucleases and pave the way for the development of specific and broad-spectrum antivirals.
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Engebretson, Jeff J., et Craig L. Moyer. « Fidelity of Select Restriction Endonucleases in Determining Microbial Diversity by Terminal-Restriction Fragment Length Polymorphism ». Applied and Environmental Microbiology 69, no 8 (août 2003) : 4823–29. http://dx.doi.org/10.1128/aem.69.8.4823-4829.2003.
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ABSTRACT An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n = 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected ≤70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).
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Tocchini-Valentini, Giuseppe D., et Glauco P. Tocchini-Valentini. « Archaeal tRNA-Splicing Endonuclease as an Effector for RNA Recombination and Novel Trans-Splicing Pathways in Eukaryotes ». Journal of Fungi 7, no 12 (12 décembre 2021) : 1069. http://dx.doi.org/10.3390/jof7121069.
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We have characterized a homodimeric tRNA endonuclease from the euryarchaeota Ferroplasma acidarmanus (FERAC), a facultative anaerobe which can grow at temperatures ranging from 35 to 42 °C. This enzyme, contrary to the eukaryal tRNA endonucleases and the homotetrameric Methanocaldococcus jannaschii (METJA) homologs, is able to cleave minimal BHB (bulge–helix–bulge) substrates at 30 °C. The expression of this enzyme in Schizosaccharomyces pombe (SCHPO) enables the use of its properties as effectors by inserting BHB motif introns into hairpin loops normally seen in mRNA transcripts. In addition, the FERAC endonuclease can create proteins with new functionalities through the recombination of protein domains.
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Bilto, Iman M., Tuhin K. Guha, Alvan Wai et Georg Hausner. « Three new active members of the I-OnuI family of homing endonucleases ». Canadian Journal of Microbiology 63, no 8 (août 2017) : 671–81. http://dx.doi.org/10.1139/cjm-2017-0067.
Texte intégralRésumé :
In vitro characterization of 3 LAGLIDADG-type homing endonucleases (HEs) (I-CcaI, I-CcaII, and I-AstI) that belong to the I-OnuI family showed that they are functional HEs that cleave their respective cognate target sites. These endonucleases are encoded within group ID introns and appear to be orthologues that have inserted into 3 different mitochondrial genes: rns, rnl, and cox3. The endonuclease activity of I-CcaI was tested using various substrates, and its minimum DNA recognition sequence was estimated to be 26 nt. This set of HEs may provide some insight into how these types of mobile elements can migrate into new locations. This study provides additional endonucleases that can be added to the catalog of currently available HEs that may have various biotechnology applications.
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Bakhrat, Anya, Melissa S. Jurica, Barry L. Stoddard et Dina Raveh. « Homology Modeling and Mutational Analysis of Ho Endonuclease of Yeast ». Genetics 166, no 2 (1 février 2004) : 721–28. http://dx.doi.org/10.1093/genetics/166.2.721.
Texte intégralRésumé :
Abstract Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.
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Turkki, Vesa, Diana Schenkwein, Oskari Timonen, Tiia Husso, Hanna P. Lesch et Seppo Ylä-Herttuala. « Lentiviral Protein Transduction with Genome-Modifying HIV-1 Integrase-I-PpoI Fusion Proteins : Studies on Specificity and Cytotoxicity ». BioMed Research International 2014 (2014) : 1–11. http://dx.doi.org/10.1155/2014/379340.
Texte intégralRésumé :
Rare-cutting endonucleases, such as the I-PpoI, can be used for the induction of double strand breaks (DSBs) in genome editing and targeted integration based on homologous recombination. For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques. For its applications, the endonuclease needs to be transported into the target cell nucleus, where the mechanism of transport may affect its function. Here, we have studied the lentiviral protein transduction of the integrase (IN)-PpoI fusion protein using thecis-packaging method. In genome-wide interaction studies, IN-fusion proteins were verified to bind their target sequence containing 28S ribosomal RNA (rRNA) genes with a 100-fold enrichment, despite the well-documented behavior of IN to be tethered into various genomic areas by host-cell factors. In addition, to estimate the applicability of the method, DSB-induced cytotoxic effects with different vector endonuclease configurations were studied in a panel of cells. Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA. In cell studies, certain cancerous cell lines were especially prone to DSBs in rRNA genes, which led us to test the protein transduction in a tumour environment in anin vivostudy. In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases.
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Shammas, Masood A., Hemanta Koley, Paola Neri, Pierfrancesco Tassone, Ramesh B. Batchu, Robert Bertheau, Robert J. Shmookler Reis et Nikhil C. Munshi. « Molecular Basis of Genomic Instability and Progression in Multiple Myeloma : Potential Role of Apurinic (Apyrimidinic) Endonuclease. » Blood 106, no 11 (16 novembre 2005) : 1561. http://dx.doi.org/10.1182/blood.v106.11.1561.1561.
Texte intégralRésumé :
Abstract Genetic instability is a prominent feature of most cancers including multiple myeloma (MM) and is responsible for ongoing accrual of mutational changes which may lead to development of drug resistance and metastasis. The molecular basis for the generation of genetic diversity in MM is therefore extremely important to understand carcinogenesis and to identify novel targets for treatment. As genomic rearrangements require excision of DNA, we hypothesized that an elevated endonuclease activity may induce recombination and subsequent genomic instability in cancer cells. We developed a plasmid degradation assay that confirmed significantly elevated endonuclease activity in MM cells compared to normal plasma cells. To identify the pre-dominating endonuclease the degradation assay was carried out in the presence of specific endonuclease inhibitors, which identified apurinic/apyrimidinic endonuclease (Ape1 and Ape2) as the predominant endonucleases in mediating increased endonuclease activity in MM. Gene expression analysis confirmed > 2 fold elevation of Ape1 or Ape2 or both in 5 of 6 MM cell lines and 12 of 15 patient samples. Both immunocytochemistry and western blot analyses confirmed upregulation of Ape1 protein in all MM cell lines and patient samples. Next, we investigated the role of elevated APE endonuclease activity in DNA recombination and subsequent genomic re-arrangements. Using a plasmid-based assay we have previously demonstrated significantly elevated homologous recombination (HR) in MM. To investigate the role of elevated AP endonuclease activity in MM, we cultured myeloma cells in the presence of methoxyamine (MX), which specifically inhibits AP endonuclease activity, and evaluated its effect on HR activity and genome-wide appearance of new mutations. Exposure of intact myeloma cells to MX resulted in > 90% inhibition of HR activity and a significant (71±10.9%; p<0.05) reduction in the appearance of new mutations compared to untreated cells, as assessed by genome-wide loss of heterozygosity (LOH) assay (Affymetrix). We also evaluated the effects of overexpression of Ape1 & 2 in normal fibroblasts which have low endonuclease activity. The transgenic upregulation of AP endonucleases (Ape1 and Ape2) in normal cells led to a significant increase in the lecombination activity, leading to a marked mutational instability as indicated by the appearance of over 20,063 and 20,143 new LOH loci per 100,000 polymorphic regions examined throughout the genome, at population doublings 25 and 50 respectively. Mutational instability was also associated with chromosomal instability confirmed by spectral karyotyping of these cells showing significant numerical and structural chromosomal abnormalities. These changes were associated with indefinite growth of cells and formation of tumors when injected in SCID mice. These data suggest that elevated AP endonuclease may be responsible for mutational and chromosomal instabilities, leading to progression of myeloma.
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Carlson, Karin, et Aud Ȗvervatin. « BACTERIOPHAGE T4 ENDONUCLEASES II AND IV, OPPOSITELY AFFECTED BY dCMP HYDROXYMETHYLASE ACTIVITY, HAVE DIFFERENT ROLES IN THE DEGRADATION AND IN THE RNA POLYMERASE-DEPENDENT REPLICATION OF T4 CYTOSINE-CONTAINING DNA ». Genetics 114, no 3 (1 novembre 1986) : 669–85. http://dx.doi.org/10.1093/genetics/114.3.669.
Texte intégralRésumé :
ABSTRACT Bacteriophage T4 mutants defective in gene 56 (dCTPase) synthesize DNA where cytosine (Cyt) partially or completely replaces hydroxymethylcytosine (HmCyt). This Cyt-DNA is degraded in vivo by T4 endonucleases II and IV, and by the exonuclease coded or controlled by genes 46 and 47.—Our results demonstrate that T4 endonuclease II is the principal enzyme initiating degradation of T4 Cyt-DNA. The activity of endonuclease IV, but not that of endonuclease II, was stimulated in the presence of a wild-type dCMP hydroxymethylase, also when no HmCyt was incorporated into phage DNA, suggesting the possibility of direct endonuclease IV-dCMP hydroxymethylase interactions. Endonuclease II activity, on the other hand, was almost completely inhibited in the presence of very small amounts of HmCyt (3-9% of total Cyt + HmCyt) in the DNA. Possible mechanisms for this inhibition are discussed.—The E. coli RNA polymerase modified by the products of T4 genes 33 and 55 was capable of initiating DNA synthesis on a Cyt-DNA template, although it probably cannot do so on an HmCyt template. In the presence of an active endonuclease IV, Cyt-DNA synthesis was arrested 10-30 min after infection, probably due to damage to the template. Cyt-DNA synthesis dependent on the unmodified (33 - 55 -) RNA polymerase was less sensitive to endonuclease IV action.
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Rahman, Md Maminur, Mohiuddin Mohiuddin, Islam Shamima Keka, Kousei Yamada, Masataka Tsuda, Hiroyuki Sasanuma, Jessica Andreani et al. « Genetic evidence for the involvement of mismatch repair proteins, PMS2 and MLH3, in a late step of homologous recombination ». Journal of Biological Chemistry 295, no 51 (2 octobre 2020) : 17460–75. http://dx.doi.org/10.1074/jbc.ra120.013521.
Texte intégralRésumé :
Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3−/− and PMS2−/− mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3DN/DN and PMS2EK/EK cells. MLH3−/− and MLH3DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme–induced double-strand breaks. PMS2−/− and PMS2EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3DN/DN and PMS2EK/EK mutations had an additive effect on the heteroallelic HR. MLH3DN/DN/PMS2EK/EK cells showed normal kinetics of γ-irradiation–induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3DN/DN/PMS2EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.
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Welch, S. G., et R. A. D. Williams. « Two thermostable type II restriction endonucleases from Icelandic strains of the genus Thermus : Tsp4C I (ACN/GT), a novel type II restriction endonuclease, and Tsp8E I, an isoschizomer of the mesophilic enzyme Bgl I (GCCNNNN/NGGC) ». Biochemical Journal 309, no 2 (15 juillet 1995) : 595–99. http://dx.doi.org/10.1042/bj3090595.
Texte intégralRésumé :
Sixteen isolates of thermophilic bacteria from the genus Thermus, isolated from neutral and alkaline hot water springs in the southwest region of Iceland, were tested for the presence of restriction endonucleases. Extracts from five of the isolates showed evidence of the presence of restriction endonuclease activity by producing discrete nucleotide fragments when incubated at 65 degrees C with lambda phage DNA. Two of the isolates (Tsp4C and Tsp8E) were found to have particularly high levels of restriction endonuclease activity, and the respective enzymes from these two Thermus isolates were partially purified and characterized and their recognition and cleavage sites were determined. Enzyme Tsp4C I is a novel Type II restriction endonuclease recognizing the interrupted palindromic tetranucleotide sequence ACNGT, where N can be any one of the four bases in DNA. Tsp4C I, which retains full enzyme activity when incubated for 10 min at temperatures up to 76 degrees C, hydrolyses the phosphodiester bond in both strands of a double-stranded DNA substrate between the third and fourth bases of the recognition sequence (ACN/GT), generating fragments with a single base 3′-OH overhang. Enzyme Tsp8E I is a thermostable isoschizomer of the mesophilic Type II restriction endonuclease Bgl I (GCCNNNN/NGGC) [Lee, Clanton and Chirikjiam (1979) Fed. Proc. 28, 294], generating fragments with a three base 3′-OH overhang. However, unlike Bgl I, Tsp8E I exhibits considerable thermal stability, retaining full enzyme activity when incubated for 10 min at temperatures up to 78 degrees C. Both Tsp4C I and Tsp8E I represent significant additions to the small but expanding list of the extremely thermostable restriction endonucleases.
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Gosálvez, J., C. López-Fernández et V. Goyanes. « Detection of cryptic bands by AluI in eukaryotic chromosomes ». Genome 32, no 4 (1 août 1989) : 672–75. http://dx.doi.org/10.1139/g89-497.
Texte intégralRésumé :
Selective digestion of fixed chromatin with the restriction endonuclease AluI (which cuts the sequence AG CT) uncovers a specific and repeatable pattern of bands within the euchromatin of two species of grasshoppers and of the L929 mouse cell line, which are not detectable by means of other banding techniques such as C-bands, specific fluorochromes, or other restriction endonucleases. It is tentatively suggested that this chromatin represents a special class of repetitive DNA embedded in the euchromatin, not containing the AluI restriction site to the same extent as in euchromatin and not associated with C-banded heterochromatic material.Key words: heterochromatin, restriction endonucleases, cytogenetics.
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Seligman, Lenny M., Kathryn M. Stephens, Jeremiah H. Savage et Raymond J. Monnat. « Genetic Analysis of the Chlamydomonas reinhadtii I-CreI Mobile Intron Homing System in Escherichia coli ». Genetics 147, no 4 (1 décembre 1997) : 1653–64. http://dx.doi.org/10.1093/genetics/147.4.1653.
Texte intégralRésumé :
Abstract We have developed and used a genetic selection system in Escherichia coli to study functional requirements for homing site recognition and cleavage by a representative eukaryotic mobile intron endonuclease. The homing endonuclease, I-CreI, was originally isolated from the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. I-CreI homing site mutants contained base pair substitutions or single base deletions that altered the rate of homing site cleavage and/or product release. I-CreI endonuclease mutants fell into six phenotypic classes that differed in in vivo activity, toxicity or genetic dominance. Inactivating mutations clustered in the N-terminal 60% of the I-CreI amino acid sequence, and two frameshift mutations were isolated that resulted in premature translation termination though retained partial activity. These mutations indicate that the N-terminal two-thirds of the I-CreI endonuclease is sufficient for homing site recognition and cleavage. Substitution mutations altered in four potential active site residues were examined D20N, Q47H or R70A substitutions inactivated endonuclease activity, whereas S22A did not. The genetic approach we have taken complements phylogenetic and structural studies of mobile intron endonucleases and has provided new information on the mechanistic basis of I-CreI homing site recognition and cleavage.
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Plessis, A., A. Perrin, J. E. Haber et B. Dujon. « Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. » Genetics 130, no 3 (1 mars 1992) : 451–60. http://dx.doi.org/10.1093/genetics/130.3.451.
Texte intégralRésumé :
Abstract The Saccharomyces cerevisiae mitochondrial endonuclease I-SceI creates a double-strand break as the initiating step in the gene conversional transfer of the omega+ intron to omega- DNA. We have expressed a galactose-inducible synthetic I-SceI gene in the nucleus of yeast that also carries the I-SceI recognition site on a plasmid substrate. We find that the galactose-induced I-SceI protein can be active in the nucleus and efficiently catalyze recombination. With a target plasmid containing direct repeats of the Escherichia coli lacZ gene, one copy of which is interrupted by a 24-bp cutting site, galactose induction produces both deletions and gene conversions. Both the kinetics and the proportion of deletions and gene conversions are very similar to analogous events initiated by a galactose-inducible HO endonuclease gene. We also find that, in a rad52 mutant strain, the repair of double-strand breaks initiated by I-SceI and by HO are similarly affected: the formation of deletions is reduced, but not eliminated. Altogether, these results suggest either that the two endonucleases act in the same way after double-strand break formation or that the two endonucleases are not involved in subsequent steps.
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Barbado, Casimiro, Dolores Córdoba-Cañero, Rafael R. Ariza et Teresa Roldán-Arjona. « Nonenzymatic release of N7-methylguanine channels repair of abasic sites into an AP endonuclease-independent pathway in Arabidopsis ». Proceedings of the National Academy of Sciences 115, no 5 (16 janvier 2018) : E916—E924. http://dx.doi.org/10.1073/pnas.1719497115.
Texte intégralRésumé :
Abasic (apurinic/apyrimidinic, AP) sites in DNA arise from spontaneous base loss or by enzymatic removal during base excision repair. It is commonly accepted that both classes of AP site have analogous biochemical properties and are equivalent substrates for AP endonucleases and AP lyases, although the relative roles of these two types of enzymes are not well understood. We provide here genetic and biochemical evidence that, in Arabidopsis, AP sites generated by spontaneous loss of N7-methylguanine (N7-meG) are exclusively repaired through an AP endonuclease-independent pathway initiated by FPG, a bifunctional DNA glycosylase with AP lyase activity. Abasic site incision catalyzed by FPG generates a single-nucleotide gap with a 3′-phosphate terminus that is processed by the DNA 3′-phosphatase ZDP before repair is completed. We further show that the major AP endonuclease in Arabidopsis (ARP) incises AP sites generated by enzymatic N7-meG excision but, unexpectedly, not those resulting from spontaneous N7-meG loss. These findings, which reveal previously undetected differences between products of enzymatic and nonenzymatic base release, may shed light on the evolution and biological roles of AP endonucleases and AP lyases.
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MAK, C. H., Y. Y. Y. CHUNG et R. C. KO. « Single-stranded endonuclease activity in the excretory–secretory products of Trichinella spiralis and Trichinella pseudospiralis ». Parasitology 120, no 5 (mai 2000) : 527–33. http://dx.doi.org/10.1017/s0031182099005879.
Texte intégralRésumé :
A novel acidic extracellular single-stranded endonuclease was demonstrated for the first time in the excretory–secretory (E–S) products of 2 species of Trichinella. Unlike the double-stranded endonuclease reported earlier, the single-stranded molecule is divalent cation independent and is detected in both T. spiralis and T. pseudospiralis E–S products. It hydrolysed single-stranded DNA and RNA at comparable rates. The single-stranded endonuclease was sensitive to inhibition by Zn2+ and to high concentrations of NaCl. Zymographic analysis indicated that it was encoded by at least 3 peptides of Mr ∼ 50–60 kDa. The rate of hydrolysis of single-stranded targets by the E–S products was substantially higher than that of the double-stranded molecule. Due to the differences in peptide profile, divalent cation dependence, and species-specific expression, the single and double-stranded endonucleases are likely to be encoded by different proteins and may have different functions.
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Tomanicek, Stephen J., Ronny C. Hughes, Joseph D. Ng et Leighton Coates. « Structure of the endonuclease IV homologue fromThermotoga maritimain the presence of active-site divalent metal ions ». Acta Crystallographica Section F Structural Biology and Crystallization Communications 66, no 9 (21 août 2010) : 1003–12. http://dx.doi.org/10.1107/s1744309110028575.
Texte intégralRésumé :
The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5′-phosphodiester bond at an AP site to generate a free 3′-hydroxyl group and a 5′-terminal sugar phosphate using their AP nuclease activity. Specifically,Thermotoga maritimaendonuclease IV is a member of the second conserved AP endonuclease family that includesEscherichia coliendonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-ray crystal structures of theT. maritimaendonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of theT. maritimaendonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily.
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Wuitschick, Jeffrey D., Paul R. Lindstrom, Alison E. Meyer et Kathleen M. Karrer. « Homing Endonucleases Encoded by Germ Line-Limited Genes in Tetrahymena thermophila Have APETELA2 DNA Binding Domains ». Eukaryotic Cell 3, no 3 (juin 2004) : 685–94. http://dx.doi.org/10.1128/ec.3.3.685-694.2004.
Texte intégralRésumé :
ABSTRACT Three insertion elements were previously found in a family of germ line-limited mobile elements, the Tlr elements, in the ciliate Tetrahymena. Each of the insertions contains an open reading frame (ORF). Sequence analysis of the deduced proteins encoded by the elements suggests that they are homing endonucleases. The genes are designated TIE1-1, TIE2-1, and TIE3-1 for Tetrahymena insertion-homing endonuclease. The endonuclease motif occupies the amino terminal half of each TIE protein. The C-terminal regions of the proteins are similar to the APETELA2 DNA binding domain of plant transcription factors. The TIE1 and TIE3 elements belong to families of repeated sequences in the germ line micronuclear genome. Comparison of the genes and the deduced proteins they encode suggests that there are at least two distinct families of homing endonuclease genes, each of which appears to be preferentially associated with a specific region of the Tlr elements. The TIE1 and TIE3 elements and their cognates undergo programmed elimination from the developing somatic macronucleus of Tetrahymena. The possible role of homing endonuclease-like genes in the DNA breakage step in developmentally programmed DNA elimination in Tetrahymena is discussed.
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Lutz, Thomas, Kiersten Flodman, Alyssa Copelas, Honorata Czapinska, Megumu Mabuchi, Alexey Fomenkov, Xinyi He, Matthias Bochtler et Shuang-yong Xu. « A protein architecture guided screen for modification dependent restriction endonucleases ». Nucleic Acids Research 47, no 18 (3 septembre 2019) : 9761–76. http://dx.doi.org/10.1093/nar/gkz755.
Texte intégralRésumé :
Abstract Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.
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Padron-Barthe, Laura, Chloé Leprêtre, Elisabeth Martin, Marie-France Counis et Alicia Torriglia. « Conformational Modification of Serpins Transforms Leukocyte Elastase Inhibitor into an Endonuclease Involved in Apoptosis ». Molecular and Cellular Biology 27, no 11 (2 avril 2007) : 4028–36. http://dx.doi.org/10.1128/mcb.01959-06.
Texte intégralRésumé :
ABSTRACT The best-characterized biochemical feature of apoptosis is degradation of genomic DNA into oligonucleosomes. The endonuclease responsible for DNA degradation in caspase-dependent apoptosis is caspase-activated DNase. In caspase-independent apoptosis, different endonucleases may be activated according to the cell line and the original insult. Among the known effectors of caspase-independent cell death, L-DNase II (LEI [leukocyte elastase inhibitor]-derived DNase II) has been previously characterized by our laboratory. We have thus shown that this endonuclease derives from the serpin superfamily member LEI by posttranslational modification (A. Torriglia, P. Perani, J. Y. Brossas, E. Chaudun, J. Treton, Y. Courtois, and M. F. Counis, Mol. Cell. Biol. 18:3612-3619, 1998). In this work, we assessed the molecular mechanism involved in the change in the enzymatic activity of this molecule from an antiprotease to an endonuclease. We report that the cleavage of LEI by elastase at its reactive center loop abolishes its antiprotease activity and leads to a conformational modification that exposes an endonuclease active site and a nuclear localization signal. This represents a novel molecular mechanism for a complete functional conversion induced by changing the conformation of a serpin. We also show that this molecular transformation affects cellular fate and that both endonuclease activity and nuclear translocation of L-DNase II are needed to induce cell death.
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Prorok, Paulina, Inga R. Grin, Bakhyt T. Matkarimov, Alexander A. Ishchenko, Jacques Laval, Dmitry O. Zharkov et Murat Saparbaev. « Evolutionary Origins of DNA Repair Pathways : Role of Oxygen Catastrophe in the Emergence of DNA Glycosylases ». Cells 10, no 7 (24 juin 2021) : 1591. http://dx.doi.org/10.3390/cells10071591.
Texte intégralRésumé :
It was proposed that the last universal common ancestor (LUCA) evolved under high temperatures in an oxygen-free environment, similar to those found in deep-sea vents and on volcanic slopes. Therefore, spontaneous DNA decay, such as base loss and cytosine deamination, was the major factor affecting LUCA’s genome integrity. Cosmic radiation due to Earth’s weak magnetic field and alkylating metabolic radicals added to these threats. Here, we propose that ancient forms of life had only two distinct repair mechanisms: versatile apurinic/apyrimidinic (AP) endonucleases to cope with both AP sites and deaminated residues, and enzymes catalyzing the direct reversal of UV and alkylation damage. The absence of uracil–DNA N-glycosylases in some Archaea, together with the presence of an AP endonuclease, which can cleave uracil-containing DNA, suggests that the AP endonuclease-initiated nucleotide incision repair (NIR) pathway evolved independently from DNA glycosylase-mediated base excision repair. NIR may be a relic that appeared in an early thermophilic ancestor to counteract spontaneous DNA damage. We hypothesize that a rise in the oxygen level in the Earth’s atmosphere ~2 Ga triggered the narrow specialization of AP endonucleases and DNA glycosylases to cope efficiently with a widened array of oxidative base damage and complex DNA lesions.
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Iino, Hitoshi, Kwang Kim, Atsuhiro Shimada, Ryoji Masui, Seiki Kuramitsu et Kenji Fukui. « Characterization of C- and N-terminal domains of Aquifex aeolicus MutL endonuclease : N-terminal domain stimulates the endonuclease activity of C-terminal domain in a zinc-dependent manner ». Bioscience Reports 31, no 5 (21 avril 2011) : 309–22. http://dx.doi.org/10.1042/bsr20100116.
Texte intégralRésumé :
DNA MMR (mismatch repair) is an excision repair system that removes mismatched bases generated primarily by failure of the 3′–5′ proofreading activity associated with replicative DNA polymerases. MutL proteins homologous to human PMS2 are the endonucleases that introduce the entry point of the excision reaction. Deficiency in PMS2 function is one of the major etiologies of hereditary non-polyposis colorectal cancers in humans. Although recent studies revealed that the CTD (C-terminal domain) of MutL harbours weak endonuclease activity, the regulatory mechanism of this activity remains unknown. In this paper, we characterize in detail the CTD and NTD (N-terminal domain) of aqMutL (Aquifex aeolicus MutL). On the one hand, CTD existed as a dimer in solution and showed weak DNA-binding and Mn2+-dependent endonuclease activities. On the other hand, NTD was monomeric and exhibited a relatively strong DNA-binding activity. It was also clarified that NTD promotes the endonuclease activity of CTD. NTD-mediated activation of CTD was abolished by depletion of the zinc-ion from the reaction mixture or by the substitution of the zinc-binding cysteine residue in CTD with an alanine. On the basis of these results, we propose a model for the intramolecular regulatory mechanism of MutL endonuclease activity.
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Coco, Rosalba Del, Liborio Stuppia, Caterina Cinti, Rita Peila et Nadir Mario Maraldi. « Ultrastructural banding induced by DraI or HaeIII progressive digestion and in situ nick translation on human chromosomes ». Genome 37, no 6 (1 décembre 1994) : 950–56. http://dx.doi.org/10.1139/g94-135.
Texte intégralRésumé :
Fixed human metaphase chromosomes were progressively digested with DraI or HaeIII restriction enzymes, submitted to in situ nick translation, and observed by transmission electron microscopy to obtain further information on the localization of the endonuclease target sequences and on the conformational changes in chromosomal bands. This approach allows us to detect specific nick translation patterns, namely, G-banding or R-like banding after short DraI and HaeIII endonuclease digestion, respectively. Intermediate banding recognizable as C-negative banding and G + C banding are induced by longer HaeIII digestion, before the C-positive banding. These patterns appear to depend both on different target sites of the employed endonucleases and on the DNA loss at different digestion times.Key words: human chromosomes, in situ nick translation, DraI banding, HaeIII banding, electron microscopy.
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Zhou, Wen, Qingtao Lu, Qingwei Li, Lei Wang, Shunhua Ding, Aihong Zhang, Xiaogang Wen, Lixin Zhang et Congming Lu. « PPR-SMR protein SOT1 has RNA endonuclease activity ». Proceedings of the National Academy of Sciences 114, no 8 (6 février 2017) : E1554—E1563. http://dx.doi.org/10.1073/pnas.1612460114.
Texte intégralRésumé :
Numerous attempts have been made to identify and engineer sequence-specific RNA endonucleases, as these would allow for efficient RNA manipulation. However, no natural RNA endonuclease that recognizes RNA in a sequence-specific manner has been described to date. Here, we report that SUPPRESSOR OF THYLAKOID FORMATION 1 (SOT1), anArabidopsispentatricopeptide repeat (PPR) protein with a small MutS-related (SMR) domain, has RNA endonuclease activity. We show that the SMR moiety of SOT1 performs the endonucleolytic maturation of 23S and 4.5S rRNA through the PPR domain, specifically recognizing a 13-nucleotide RNA sequence in the 5′ end of the chloroplast 23S–4.5S rRNA precursor. In addition, we successfully engineered the SOT1 protein with altered PPR motifs to recognize and cleave a predicted RNA substrate. Our findings point to SOT1 as an exciting tool for RNA manipulation.
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Xu, Shuang-yong, et Yogesh K. Gupta. « Natural zinc ribbon HNH endonucleases and engineered zinc finger nicking endonuclease ». Nucleic Acids Research 41, no 1 (2 novembre 2012) : 378–90. http://dx.doi.org/10.1093/nar/gks1043.
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Gnügge, Robert, et Lorraine S. Symington. « Efficient DNA double-strand break formation at single or multiple defined sites in the Saccharomyces cerevisiae genome ». Nucleic Acids Research 48, no 20 (14 octobre 2020) : e115-e115. http://dx.doi.org/10.1093/nar/gkaa833.
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Abstract DNA double-strand breaks (DSBs) are common genome lesions that threaten genome stability and cell survival. Cells use sophisticated repair machineries to detect and heal DSBs. To study DSB repair pathways and associated factors, inducible site-specific endonucleases have proven to be fundamental tools. In Saccharomyces cerevisiae, galactose-inducible rare-cutting endonucleases are commonly used to create a single DSB at a unique cleavage site. Galactose induction requires cell cultivation in suboptimal growth media, which is tedious especially when working with slow growing DSB repair mutants. Moreover, endonucleases that simultaneously create DSBs in multiple defined and unique loci of the yeast genome are not available, hindering studies of DSB repair in different genomic regions and chromatin contexts. Here, we present new tools to overcome these limitations. We employ a heterologous media-independent induction system to express the yeast HO endonuclease or bacterial restriction enzymes for single or multiple DSB formation, respectively. The systems facilitate tightly controlled and efficient DSB formation at defined genomic sites and will be valuable tools to study DSB repair at a local and genome-wide scale.
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von Kanel, Thomas, Dominik Gerber, André Schaller, Alessandra Baumer, Eva Wey, Christopher B. Jackson, Franziska M. Gisler, Karl Heinimann et Sabina Gallati. « Quantitative 1-Step DNA Methylation Analysis with Native Genomic DNA as Template ». Clinical Chemistry 56, no 7 (1 juillet 2010) : 1098–106. http://dx.doi.org/10.1373/clinchem.2009.142828.
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Abstract Background: DNA methylation analysis currently requires complex multistep procedures based on bisulfite conversion of unmethylated cytosines or on methylation-sensitive endonucleases. To facilitate DNA methylation analysis, we have developed a quantitative 1-step assay for DNA methylation analysis. Methods: The assay is based on combining methylation-sensitive FastDigest® endonuclease digestion and quantitative real-time PCR (qPCR) in a single reaction. The first step consists of DNA digestion, followed by endonuclease inactivation and qPCR. The degree of DNA methylation is evaluated by comparing the quantification cycles of a reaction containing a methylation-sensitive endonuclease with the reaction of a sham mixture containing no endonuclease. Control reactions interrogating an unmethylated locus allow the detection and correction of artifacts caused by endonuclease inhibitors, while simultaneously permitting copy number assessment of the locus of interest. Results: With our novel approach, we correctly diagnosed the imprinting disorders Prader–Willi syndrome and Angelman syndrome in 35 individuals by measuring methylation levels and copy numbers for the SNRPN (small nuclear ribonucleoprotein polypeptide N) promoter. We also demonstrated that the proposed correction model significantly (P < 0.05) increases the assay’s accuracy with low-quality DNA, allowing analysis of DNA samples with decreased digestibility, as is often the case in retrospective studies. Conclusions: Our novel DNA methylation assay reduces both the hands-on time and errors caused by handling and pipetting and allows methylation analyses to be completed within 90 min after DNA extraction. Combined with its precision and reliability, these features make the assay well suited for diagnostic procedures as well as high-throughput analyses.
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Kwiatek, Agnieszka, Maciej Łuczkiewicz, Katarzyna Bandyra, Daniel C. Stein et Andrzej Piekarowicz. « Neisseria gonorrhoeae FA1090 Carries Genes Encoding Two Classes of Vsr Endonucleases ». Journal of Bacteriology 192, no 15 (28 mai 2010) : 3951–60. http://dx.doi.org/10.1128/jb.00098-10.
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ABSTRACT A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases V.NgoAXIII and V.NgoAXIV from Neisseria gonorrhoeae FA1090 based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli, the proteins were biochemically characterized and the endonucleolytic activities and specificities of V.NgoAXIII and V.NgoAXIV were determined. V.NgoAXIII was found to be multispecific and to recognize T:G mismatches in every nucleotide context tested, whereas V.NgoAXIV recognized T:G mismatches in the following sequences: GTGG, CTGG, GTGC, ATGC, and CTGC. Alanine mutagenesis of conserved residues showed that Asp50 and His68 of V.NgoAXIII and Asp51 and His69 of V.NgoAXIV are essential for hydrolytic activity. Glu25, His64, and Asp97 of V.NgoAXIV and Glu24, Asp63, and Asp97 of V.NgoAXIII are important but not crucial for the activity of V.NgoAXIII and V.NgoAXIV. However, Glu24 and Asp63 are also important for the specificity of V.NgoAXIII. On the basis of our results concerning features of Vsr endonucleases expressed by N. gonorrhoeae FA1090, we postulate that at least two types of Vsr endonucleases can be distinguished.
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Batistatou, A., et L. A. Greene. « Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation : correlation with suppression of endonuclease activity. » Journal of Cell Biology 115, no 2 (15 octobre 1991) : 461–71. http://dx.doi.org/10.1083/jcb.115.2.461.
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Past studies have shown that serum-free cultures of PC12 cells are a useful model system for studying the neuronal cell death which occurs after neurotrophic factor deprivation. In this experimental paradigm, nerve growth factor (NGF) rescues the cells from death. It is reported here that serum-deprived PC12 cells manifest an endonuclease activity that leads to internucleosomal cleavage of their cellular DNA. This activity is detected within 3 h of serum withdrawal and several hours before any morphological sign of cell degeneration or death. NGF and serum, which promote survival of the cells, inhibit the DNA fragmentation. Aurintricarboxylic acid (ATA), a general inhibitor of nucleases in vitro, suppresses the endonuclease activity and promotes long-term survival of PC12 cells in serum-free cultures. This effect appears to be independent of macromolecular synthesis. In addition, ATA promotes long-term survival of cultured sympathetic neurons after NGF withdrawal. ATA neither promotes nor maintains neurite outgrowth. It is hypothesized that the activation of an endogenous endonuclease could be responsible for neuronal cell death after neurotrophic factor deprivation and that growth factors could promote survival by leading to inhibition of constitutively present endonucleases.
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Zhou, Xiaohong, Maria DeLucia et Jinwoo Ahn. « SLX4-SLX1 Protein-independent Down-regulation of MUS81-EME1 Protein by HIV-1 Viral Protein R (Vpr) ». Journal of Biological Chemistry 291, no 33 (27 juin 2016) : 16936–47. http://dx.doi.org/10.1074/jbc.m116.721183.
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Evolutionarily conserved structure-selective endonuclease MUS81 forms a complex with EME1 and further associates with another endonuclease SLX4-SLX1 to form a four-subunit complex of MUS81-EME1-SLX4-SLX1, coordinating distinctive biochemical activities of both endonucleases in DNA repair. Viral protein R (Vpr), a highly conserved accessory protein in primate lentiviruses, was previously reported to bind SLX4 to mediate down-regulation of MUS81. However, the detailed mechanism underlying MUS81 down-regulation is unclear. Here, we report that HIV-1 Vpr down-regulates both MUS81 and its cofactor EME1 by hijacking the host CRL4-DCAF1 E3 ubiquitin ligase. Multiple Vpr variants, from HIV-1 and SIV, down-regulate both MUS81 and EME1. Furthermore, a C-terminally truncated Vpr mutant and point mutants R80A and Q65R, all of which lack G2 arrest activity, are able to down-regulate MUS81-EME1, suggesting that Vpr-induced G2 arrest is not correlated with MUS81-EME1 down-regulation. We also show that neither the interaction of MUS81-EME1 with Vpr nor their down-regulation is dependent on SLX4-SLX1. Together, these data provide new insight on a conserved function of Vpr in a host endonuclease down-regulation.
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Liu, Guang, Zhenyi Zhang, Gong Zhao, Zixin Deng, Geng Wu et Xinyi He. « Crystallization and preliminary X-ray analysis of the type IV restriction endonuclease ScoMcrA fromStreptomyces coelicolor, which cleaves both Dcm-methylated DNA and phosphorothioated DNA ». Acta Crystallographica Section F Structural Biology Communications 71, no 1 (1 janvier 2015) : 57–60. http://dx.doi.org/10.1107/s2053230x14025801.
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ScoMcrA is a type IV modification-dependent restriction endonuclease found in the model strainStreptomyces coelicolor. Unlike type I, II and III restriction endonucleases, which cleave unmodified DNA, type IV restriction endonucleases cleave modified DNA, including methylated, hydroxymethylated, glucosyl-hydroxymethylated and phosphorothioated DNA. ScoMcrA targets both Dcm-methylated DNA and phosphorothioated DNA, and makes double-strand breaks 16–28 nt away from the modified nucleotides or the phosphorothioate links. However, the mechanism by which ScoMcrA recognizes these two entirely different types of modification remains unclear. In this study, the ScoMcrA protein was overexpressed, purified and crystallized. The crystals diffracted to 3.35 Å resolution and belonged to space groupP212121. The unit-cell parameters were determined to bea= 130.19,b= 139.36,c= 281.01 Å, α = β = γ = 90°. These results will facilitate the detailed structural analysis of ScoMcrA and further elucidation of its biochemical mechanism.
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Li, Zhuokun, Xiaojue Wang, Dongyang Xu, Dengwei Zhang, Dan Wang, Xuechen Dai, Qi Wang et al. « DNB-based on-chip motif finding : A high-throughput method to profile different types of protein-DNA interactions ». Science Advances 6, no 31 (juillet 2020) : eabb3350. http://dx.doi.org/10.1126/sciadv.abb3350.
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Here, we report a sensitive DocMF system that uses next-generation sequencing chips to profile protein-DNA interactions. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer adjacent motif (PAM) sequences of different CRISPR systems. DocMF can simultaneously screen both 5′ and 3′ PAMs with high coverage. For SpCas9, we found noncanonical 5′-NAG-3′ (~5%) and 5′-NGA-3′ (~1.6%), in addition to its common PAMs, 5′-NGG-3′ (~89.9%). More relaxed PAM sequences of two uncharacterized Cas endonucleases, VeCas9 and BvCas12a, were extensively characterized using DocMF. Moreover, we observed that dCas9, a DNA binding protein lacking endonuclease activity, preferably bound to the previously reported 5′-NGG-3′ sequence. In summary, our studies demonstrate that DocMF is the first tool with the capacity to exhaustively assay both the binding and the cutting properties of different DNA binding proteins.
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Hill, A. W., et J. A. Leigh. « DNA fingerprinting ofStreptococcus uberis : a useful tool for epidemiology of bovine mastitis ». Epidemiology and Infection 103, no 1 (août 1989) : 165–71. http://dx.doi.org/10.1017/s0950268800030466.
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SUMMARYA simple and reproducible typing system based on restriction fragment size of chromosomal DNA was developed to compare isolates ofStreptococcus uberisobtained from the bovine mammary gland. The endonuclease giving the most useful restriction patterns wasHindIII, although seven other endonucleases (Bgl1,EcoR1,Not1,Pst1,Sfi1,Sma1,Xba1) were also tested in the system. An image analyser was used to obtain a densitometric scan and a graphic display of the restriction patterns. Such a system will allow large scale data storage for future computer-aided comparison.
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Basnakian, Alexei G., Norishi Ueda, Gur P. Kaushal, Marina V. Mikhailova et Sudhir V. Shah. « DNase I-Like Endonuclease in Rat Kidney Cortex That Is Activated during Ischemia/Reperfusion Injury ». Journal of the American Society of Nephrology 13, no 4 (avril 2002) : 1000–1007. http://dx.doi.org/10.1681/asn.v1341000.
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ABSTRACT. Ischemia/reperfusion is known to result in DNA fragmentation and cell death in kidney tubular epithelium, but the endonucleases responsible for this DNA damage have not been identified. DNA substrate gel analysis of extracts from normal rat kidney cortex revealed the presence of a DNase with an apparent molecular mass of 30 to 34 kD. This enzyme is not a dimer of the previously described nuclear 15-kD endonuclease in kidney cells. Partially purified DNase exhibited characteristics similar to those of rat DNase I. The DNase was able to digest circular DNA (endonuclease), required both Ca2+ and Mg2+ ions, and was inhibited by Zn2+ and by aurintricarboxylic acid; it was not inhibited by G-actin. Rat kidneys were subjected to 40 min of ischemia, followed by 0, 1, 4, 16, or 48 h of reperfusion. The activity of the DNase in cytosolic and nuclear extracts, the 200-bp ladder-generating activity, and 3′OH strand breaks in nuclear DNA were simultaneously increased after ischemia, during the first hours of reperfusion. Oxidative DNA damage, measured as 8-hydroxydeoxyguanosine content, did not coincide with endonuclease-generated DNA breaks. Oxidative DNA damage was increased during ischemia and gradually decreased during reperfusion. Phosphorothioated DNase I antisense oligodeoxynucleotide introduced into cultured NRK-52E rat kidney epithelial cells inhibited DNA fragmentation and attenuated cell death induced by hypoxia/reoxygenation in vitro. The data indicate that the DNase I-like endonuclease may contribute to DNA fragmentation in reperfused rat kidneys.