SEARCH · PubMed Health

Results for “Endonucleases”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources

PubMed2004

Bsu2413I and Bfi2411I, two new thermophilic type II restriction endonucleases from Bacillus subtilis and Bacillus firmus: isolation and partial purification. Thermophilic endonucleases from two Bacillus species.

Two new thermophilic type II restriction endonucleases, which we designated as Bsu2413I and Bfi2411I, have been isolated from gram-positive thermophilic bacteria Bacillus subtilis strain 2413 and Bacillus firmus strain 2411 respectively and partially purified. The restriction endonucleases were extracted from cell extracts and purified using single step purification through phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights of 33 and 67 kDa for the Bsu2413I and 39 and 67 kDa for the Bfi2411I. The partially purified Bsu2413I enzyme restricted pBR322 DNA into two fragments of 3250 and 1100 bp whereas Bfi2411I enzyme restricted pBR322 DNA into two fragments of 3500 and 800 bp. The activity of both endonucleases was assayed at 55 degrees C and they required Mg+2 as cofactor like other type II restriction endonucleases.

Bacillus↗

PubMed2001

Regulation of DNAS1L3 endonuclease activity by poly(ADP-ribosyl)ation during etoposide-induced apoptosis. Role of poly(ADP-ribose) polymerase-1 cleavage in endonuclease activation.

Several endonucleases are implicated in the internucleosomal DNA fragmentation associated with apoptosis. The human Ca2+- and Mg2+-dependent endonuclease DNAS1L3 is inhibited by poly(ADP-ribosyl)ation in vitro, and its activation during apoptosis shows a time course similar to that of the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The role of the cleavage and consequent inactivation of PARP-1 by caspase-3 in the activation of DNAS1L3 has now been investigated further both in vitro and in vivo. In an in vitro system based on purified recombinant proteins and NAD, caspase-3 prevented the inhibition of DNAS1L3 endonuclease activity by wild-type PARP-1 but not that induced by a caspase-3-resistant PARP-1 mutant. The induction by etoposide of apoptosis in human osteosarcoma cells (which were shown not to express endogenous DNAS1L3) was accompanied by internucleosomal DNA fragmentation only after transfection of the cells with a plasmid encoding DNAS1L3. This DNA fragmentation in etoposide-treated cells was blocked by 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, an inhibitor of intracellular Ca2+ release. Expression of the endonuclease subunit of DNA fragmentation factor (DFF40) and cleavage of its inhibitor, DFF45, were not sufficient to cause internucleosomal DNA fragmentation in osteosarcoma cells during etoposide-induced apoptosis. Coexpression of caspase-3-resistant PARP-1 mutant with DNAS1L3 in osteosarcoma cells blocked etoposide-induced internucleosomal DNA fragmentation and resulted in persistent poly(ADP-ribosyl)ation of DNAS1L3; it did not, however, prevent the activation of caspase-3 and the consequent cleavage of endogenous PARP-1. These results indicate that PARP-1 cleavage during apoptosis is not simply required to prevent excessive depletion of NAD and ATP but is also necessary to release DNAS1L3 from poly(ADP-ribosyl)ation-mediated inhibition.

Antineoplastic Agents, Phytogenic↗

PubMed2002

Intronless homing: site-specific endonuclease SegF of bacteriophage T4 mediates localized marker exclusion analogous to homing endonucleases of group I introns.

All genetic markers from phage T2 are partially excluded from the progeny of mixed infections with the related phage T4 (general, or phage exclusion). Several loci, including gene 56 of T2, are more dramatically excluded, being present in only approximately 1% of the progeny. This phenomenon is referred to as localized marker exclusion. Gene 69 is adjacent to gene 56 of T4 but is absent in T2, being replaced by completely nonhomologous DNA. We describe SegF, a novel site-specific DNA endonuclease encoded by gene 69, which is similar to GIY-YIG homing endonucleases of group I introns. Interestingly, SegF preferentially cleaves gene 56 of T2, both in vitro and in vivo, compared with that of phage T4. Repair of the double-strand break (DSB) results in the predominance of T4 genes 56 and segF in the progeny, with exclusion of the corresponding T2 sequences. Localized exclusion of T2 gene 56 is dependent on full-length SegF and is likely analogous to group I intron homing, in which repair of a DSB results in coconversion of markers in the flanking DNA. Phage T4 has many optional homing endonuclease genes similar to segF, whereas similar endonuclease genes are relatively rare in other members of the T-even family of bacteriophages. We propose that the general advantage enjoyed by T4 phage, over almost all of its relatives, is a cumulative effect of many of these localized events.

Amino Acid Sequence↗

PubMed1976

Endonucleases and simian virus 40 virions: origin of a virion-associated endonuclease.

The origin and role of the endonuclease activity associated with purified virions of simian virus 40, previously described by this and other laboratories, have been further investigated. We found that the enzymatic activity from virions of temperature-sensitive (ts) mutants is not more heat labile than that from wild-type virions. This result was obtained for a variety of ts mutants, including three of the tsA class, and in experiments in which the enzyme was tested in both the presence and absence of viral particles. Comparison of the viron enzyme with endonucleases prepared from either serum or nuclei of uninfected cells reveals a similarity between the viron and serum enzymes based on chromatographic behavior and relative activity with different cations. Virus particles prepared free of this endonuclease were still infectious. We were unsuccessful in uncovering endonuclease in such preparations upon disruption. These data emphasize the necessity for caution in interpreting the role of particle-associated enzymes.

Animals↗

PubMed1999

REF Select: expert system software for selecting restriction endonucleases for restriction endonuclease fingerprinting.

REF Select, expert system software, has been developed to assist in the selection of optimal restriction endonucleases for restriction endonuclease fingerprinting (REF), a method for rapid and sensitive mutation screening of long DNA segments (1-2 kb). The REF method typically involves six separate digestions with up to two restriction endnonucleases used in each digestion. If done manually, performing a comprehensive review of the large number of possible sets of restriction endonucleases that could be used (over 10(19) in the example presented here) and making an optimal choice is not feasible. Furthermore, the typical nonoptimal manual selection takes approximately 8 h by someone experienced with REF. REF Select enables a comprehensive review of the possible sets and a consistent, objective and fast selection of an optimal set by using a two-step strategy: the selection of sets that meet specific constraints, which is followed by a ranking of those sets by an optimality score. Based on our experience with REF, we chose default selection and ranking parameters to help the user get started quickly. These parameters form a knowledge base that can be customized and then saved by the user. In conclusion, REF Select facilitates the general application of REF by serving as an expert system for the selection of optimal restriction endonucleases. We demonstrated REF Select using an example segment from the human p53 gene.

Algorithms↗

PubMed1990

Studies on rat liver nuclear DNA damaged by chemical carcinogen (3'-Me DAB) and AP DNA endonuclease. II. Kinetic properties of AP DNA endonucleases in rat liver chromatin.

An experiment was designed to investigate the reaction mechanism of AP (apurinic or apyrimidinic) DNA endonucleases (APcI, APcII, APcIII) purified from rat liver chromatin. Sulfhydryl compounds (2-mercaptoethanol, dithiothreitol) brought about optimal activities of AP DNA endonucleases and N-ethylmaleimide or HgCl2 inhibited the enzyme activities, indicating the presence of sulfhydryl group at or near the active sites of the enzymes. Mg2+ was essential and 4mM of Mg2+ was sufficient for the optimal activities of AP DNA endonucleases. Km values of APcI, APcII and APcIII for the substrate (E. coli chromosomal AP DNA) were 0.53, 0.27 and 0.36 microM AP sites, respectively. AMP was the most potent inhibitor among adenine nucleotides tested and the inhibition was uncompetitive with respective to the substrate. The Ki values of APcI, APcII and APcIII were 0.35, 0.54 and 0.41mM, respectively. The degree of nick translation of AP DNAs nicked by APcI, APcII and APcIII with Klenow fragment in the presence and absence of T4 polynucleotide kinase or alkaline phosphatase were the same, suggesting that all 3 AP DNA endonucleases excise the phosphodiester bond of AP DNA strand to release 3-hydroxyl nucleotides and 5-phosphomonoester nucleotides.

Animals↗

PubMed1999

Endonuclease III and endonuclease IV protect Escherichia coli from the lethal and mutagenic effects of near-UV irradiation.

In contrast to the DNA damage caused by far-UV (lambda < 290 nm), near-UV (290 < lambda < 400 nm) induced DNA damage is partially oxygen dependent, suggesting the involvement of reactive oxygen species. To test the hypothesis that enzymes that protect cells from oxidative DNA damage are also involved in preventing near-UV mediated DNA damage, isogenic strains deficient in one or more of exonuclease III (xthA), endonuclease IV (nfo), and endonuclease III (nth) were exposed to increasing levels of far-UV and near-UV. All strains, with the exception of the nth single mutant, were found to be hypersensitive to the lethal effects of near-UV relative to a wild-type strain. A triple mutant strain (nth nfo xthA) exhibited the greatest sensitivity to near-UV-mediated lethality. The triple mutant was more sensitive than the nfo xthA double mutant to the lethal effects of near-UV, but not far-UV. A forward mutation assay also revealed a significantly increased sensitivity for the triple mutant compared to the nfo xthA deficient strain in the presence of near-UV. However, the triple mutant was no more sensitive to the mutagenic effects of far-UV than a nfo xthA double mutant. These data suggest that exonuclease III, endonuclease IV, and endonuclease III are important in protection against near-UV-induced DNA damage.

Carbon-Oxygen Lyases↗

PubMed2001

A model of structure and action of Sau3AI restriction endonuclease that comprises two MutH-like endonuclease domains within a single polypeptide.

Sau3AI is a type II endonuclease that cleaves GATC sequences, producing sticky ends with 4-nucleotide 5'-overhangs. Its activity is inhibited by cytosine C5-methylation within the target sequence. In the N-terminus, Sau3AI exhibits sequence similarity to the GATC-specific single-strand nicking endonuclease MutH implicated in mismatch repair (Ban and Yang, 1998). Sequence analysis of Sau3AI and its homologs reveals that Sau3AI possesses an additional MutH-like domain in the C-terminus. Structure prediction suggests that the C-terminal domain lacks the endonuclease active site but retains all putative DNA-binding elements. As an illustration of these findings, a model of quaternary structure of Sau3AI complexed with the target DNA is presented. These predictions have implications for evolution, structure and function of bacterial DNA repair enzymes and restriction endonucleases.

Amino Acid Sequence↗

PubMed1991

The highly homologous isoschizomers RsrI endonuclease and EcoRI endonuclease do not recognize their target sequence identically.

Using a series of decadeoxyribonucleotides containing base analogues as substrates we measured the steady-state kinetic parameters for the reaction catalyzed by RsrI endonuclease and compared the results to those with its isoschizomer EcoRI. The kinetics of RsrI cleavage are affected by each substitution, with the effects being generally more deleterious than with EcoRI, as shown by the greater reduction in the specificity constant kcat/KM. The magnitudes of the effects of several substitutions are consistent with the formation of direct enzyme-nucleobase contacts at the indicated positions. With substrates containing 2-amino-purine or 2,6-diaminopurine at the central adenine or uracil at the outermost thymine in the recognition sequence, cleavage by RsrI was very slow, less than one-tenth the rate of the corresponding EcoRI-catalyzed reaction. The lower tolerance of RsrI endonuclease for functional group changes in its recognition site may reflect differences in the mechanisms of DNA recognition by the two enzymes. Although RsrI and EcoRI endonucleases bind with similar affinities to specific and nonspecific DNA sequences and appear to introduce similar structural distortions in DNA upon binding, the use of substrate analogues reveals significant differences at the level of catalysis in the mechanisms by which these two endonucleases recognize the duplex sequence GAATTC.

Base Sequence↗

PubMed1975

Endonuclease III: an endonuclease from Escherichia coli that introduces single polynucleotide chain scissions in ultraviolet-irradiated DNA.

An endonuclease that makes single polynucleotide chain scissions in UV-irradiated DNA has been purified from Escherichia coli. The activity has the following properties: (1) unirradiated DNA is attacked very little if at all; (2) single-stranded DNA is not attacked, whether irradiated or not; (3) there is no requirement for divalent cations, and the activity is not affected by addition of EDTA; (4) the pH optimum is approximately 7; (5) the activity is inhibited by 1 M NaCl, single-stranded DNA, transfer RNA, and unirradiated double-stranded DNA; (6) the sedimentation coefficient, S20, W, is approximately 2.6; (7) it is a basic protein. The enzyme is tentatively named E. coli endonuclease III. The physiological function of the endonuclease has not yet been established.

Centrifugation, Density Gradient↗

PubMed1975

Host DNA degradation after infection of Escherichia coli with bacteriophage T4: dependence of the alternate pathway of degradation which occurs in the absence of both T4 endonuclease II and nuclear disruption on T4 endonuclease IV.

Escherichia coli cells infected with T4 phage which are deficient in both nuclear disruption and endonuclease II exhibit a pathway of host DNA degradation which does not occur in cells infected with phage deficient only in endonuclease II. This alternate pathway of host DNA degradation requires T4 endonuclease IV.

Cell Nucleus↗

PubMed1997

A 55-kDa endonuclease of mammalian mitochondria: comparison of its subcellular localization and endonucleolytic properties with those of endonuclease G.

A novel endonuclease of 55-kDa was found in rat liver mitochondria by a zymographic assay, in addition to the 29 kDa enzyme that is well-known as endonuclease G (Endo G). Subcellular localization of these enzymes in rat liver cells was examined by biochemical fractionation. Endo G was located in both nuclei and mitochondria as has been previously reported, while the 55-kDa enzyme was only detected in the mitochondrial fraction. The levels of the endonucleases in the mitochondria varied greatly among the rat organs, and the activity in the heart was about 30 times higher than that in the liver. The 55-kDa enzyme and Endo G were extracted from bovine heart mitochondria with 0.4 M NaCl. During purification the 55-kDa enzyme and Endo G were copurified because of their similar chromatographic behavior, so they were separated by gel filtration or electrophoresis in the presence of SDS and the proteins were then renatured. The nucleolytic properties of the 55-kDa enzyme resembled those of Endo G and other known mitochondrial nucleases. The enzyme degraded single-stranded DNA more rapidly than duplex DNA at a weak alkaline pH1 requiring Mg2+ or Mn2+ but not Ca2+ or Zn2+. Nicks generated by the enzyme had 5'-P and 3'-OH ends. The 55-kDa enzyme, like Endo G, displayed an unusually strong preference to nick within a (dG)n.(dC)n tract.

Animals↗

PubMed1987

Thymine glycol-DNA glycosylase/AP endonuclease of CEM-C1 lymphoblasts: a human analog of Escherichia coli endonuclease III.

A thymine glycol-DNA glycosylase/AP endonuclease has been identified in human CEM-C1 lymphoblasts. The enzyme is active in the absence of divalent cations and has an apparent molecular size of approximately 60,000 daltons. The enzyme releases thymine glycol from osmium tetroxide-damaged DNA via an N-glycosylase activity and is associated with an endonuclease activity that mediates phosphodiester bond cleavage at sites of thymine glycol and apurinic sites. We propose that this enzyme, which we call redoxyendonuclease, is the human analog of a bacterial enzyme, E. coli endonuclease III, that recognizes oxidative DNA damage.

Cations, Divalent↗

PubMed1985

Identification and initial characterisation of a pyrimidine dimer UV endonuclease (UV endonuclease beta) from Deinococcus radiodurans; a DNA-repair enzyme that requires manganese ions.

An endonuclease that incises lightly ultraviolet-irradiated supercoiled plasmid DNA was identified in cell-free extracts of Deinococcus radiodurans R1 wild-type. The endonuclease was absent from strains mutant in the uvsC, uvsD or uvsE genes identifying it as 'UV endonuclease beta' responsible for the initial incision step of one excision-repair pathway for the removal of pyrimidine dimers from D. radiodurans DNA in vivo. The enzyme was purified free from contaminating nuclease activities and was partially characterised. The enzyme has an apparent molecular weight of 36 000, is ATP-independent, caffeine-insensitive and is inactivated by N-ethylmaleimide. It also has a novel requirement for manganese ions distinguishing it from all other known DNA-repair enzymes.

Chromatography, Gel↗

PubMed1998

Endonuclease II of coliphage T4: a recombinase disguised as a restriction endonuclease?

EndoII shares with restriction endonucleases the property of cleaving foreign DNA while leaving the endonuclease-encoding genome intact, ensuring the survival of one DNA species in the cell. In addition, in vivo EndoII cleaves a specific DNA sequence and cleavage is context dependent. These context effects extend over at least 1000 bp, largely limiting cleavage to once within this distance. Like homing endonucleases, in vivo EndoII recognizes a long, asymmetric and degenerate consensus sequence which has two distinct parts. Recognition of one part of the consensus sequence involves base-specific bonds, and recognition of the other involves sequence-dependent helical structure. EndoII fulfills an obvious short-term survival role in ensuring the dominance of phage DNA in an infected cell, but may also have a long-term evolutionary role, producing gene-size fragments of foreign DNA to be enrolled in the phage genetic repertoire.

Bacteriophage T4↗

PubMed1998

Deletion analysis of human AP-endonuclease: minimum sequence required for the endonuclease activity.

AP-endonuclease (APE) plays a central role in the base excision repair process for oxidative and alkylation damage in DNA. The major human APE with 318 amino acid residues possesses transcriptional regulatory activities for which the N-terminal region was found to be essential. Systematic deletion studies of both amino and carboxyl termini of the human APE have shown that the carboxyl termini residues are essential for the endonuclease activity as indicated by direct measurement of enzyme activity and from studies on phenotypic rescue of Escherichia coli. However, the amino-terminal residues are dispensable for this activity and the boundary of the active endonuclease lies at this end between positions 61 and 80.

Carbon-Oxygen Lyases↗

PubMed2005

A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease.

A stable heterodimeric protein containing a single correctly folded catalytic domain (SCD) of T7 endonuclease I was produced by means of a trans-splicing intein system. As predicted by a model presented earlier, purified SCD protein acts a non-specific nicking endonuclease on normal linear DNA. The SCD retains some ability to recognize and cleave a deviated DNA double-helix near a nick or a strand-crossing site. Thus, we infer that the non-specific and nicked-site cleavage activities observed for the native T7 endonuclease I (as distinct from the resolution activity) are due to uncoordinated actions of the catalytic domains. The positively charged C-terminus of T7 Endo I is essential for the enzymatic activity of SCD, as it is for the native enzyme. We propose that the preference of the native enzyme for the resolution reaction is achieved by cooperativity in the binding of its two catalytic domains when presented with two of the arms across a four-way junction or cruciform structure.

Bacteriophage T7↗

PubMed1990

Purification and characterization of HeLa endonuclease R. A G-specific mammalian endonuclease.

We previously reported a double-stranded endonuclease from HeLa cells, endonuclease R (endo R), which specifically cleaves duplex DNA at sites rich in G.C base pairs. In this report we describe the purification of endo R to near homogeneity by conventional and affinity chromatography. The molecular mass of the active form of endo R is approximately 115-125 kDa. SDS-gel electrophoresis reveals a major protein species of 100 kDa. The enzyme requires Mg2+ as a cofactor and is equally active on closed circular and linear duplex DNA substrates that contain G-rich sequences. A 50% reduction in cleavage activity is observed with Ca2+ ions and no double-stranded cleavage occurs with Zn2+. Use of Mn2+ causes an altered specificity at low concentrations of enzyme or divalent metal ion and nonspecific degradation of the substrate at higher concentrations. Endo R is strongly inhibited by sodium or potassium chloride and exhibits a wide pH optimum of 6.0-9.0. The pI of the enzyme is between 6.5 and 7.0. A 2-fold stimulation is observed with the addition of dGTP or dATP but specific cleavage is inhibited by ATP at an equivalent concentration. Cleavage activity is competitively inhibited 10-fold more efficiently by single-stranded poly(dG)12 than by other DNA competitors. The ends of endo R cleavage products contain 5'-phosphate and 3'-hydroxyl groups, and a significant portion of these products were substrates for T4 DNA ligase. Endo R appears to be a previously uncharacterized mammalian endonuclease.

Base Composition↗