Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DNase VIII is an exonuclease purified from human placenta trophoblast nuclei. The enzyme has a pH optimum of 9.5 and requires a divalent cation. It is inhibited by salt and stimulated by Triton X-100. Glycerol gradient analysis of the activity indicates a sedimentation coefficient of 2.8 S (31,000 daltons if globular). This enzyme initiates hydrolysis from 5'-phosphorylated termini of single-stranded DNA and acts at internal phosphodiester bonds liberating 5'-phosphorylated oligonucleotides. It degrades polynucleotides of repeating base sequence as well as single-stranded DNA, yielding oligonucleotides of even number, in which the main reaction products are dinucleotides. The activity on denatured DNA is not inhibited by the presence of ultraviolet-induced photoproducts. DNase VIII can also initiate hydrolysis at those distorted termini produced by the action of Micrococcus luteus dimer specific endonuclease on duplex DNA, which contains cyclobutane dimers.
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PMID:Purification and characterization of DNase VIII. A 5'-3' directed exonuclease from human placental nuclei. 682 22

Oligonucleotides containing a unique alpha-deoxyadenosine or tetrahydrofuran (a model abasic site) were synthesized using phosphoramidite chemistry. Repair enzymes from Escherichia coli, including endonucleases III, IV, and VIII, exonuclease III, formamidopyrimidine N-glycosylase, and deoxyinosine 3'-endonuclease, as well as UV dimer N-glycosylases from T4 (den V) and Micrococcus luteus, were examined for their ability to recognize alpha-deoxyadenosine and tetrahydrofuran. In agreement with prior studies, a tetrahydrofuran-containing oligonucleotide was a substrate for endonuclease IV and exonuclease III, but not for the other repair enzymes. However, an oligonucleotide containing alpha-deoxyadenine was a substrate only for endonuclease IV. Competitive inhibition studies with both substrates confirmed that the activity recognizing alpha-deoxyadenine was endonuclease IV and not a possible contaminant in the endonuclease IV preparation. Using E. coli extracts, the activity that recognized alpha-deoxyadenine was dependent on nfo, the structural gene of endonuclease IV, further substantiating that endonuclease IV is the enzyme that recognized alpha-deoxyadenine. Kinetic measurements indicated that alpha-deoxyadenosine was as good a substrate for endonuclease IV as tetrahydrofuran; the Km and Vmax values for both substrates were similar. Using substrates that were labeled at either the 3'- or 5'-terminus, endonuclease IV was shown to hydrolyze the phosphodiester bond 5' to either alpha-deoxyadenosine or tetrahydrofuran, leaving the lesion, alpha-deoxyadenosine or tetrahydrofuran, on the 5'-terminus of the nicked site. The ability of endonuclease IV to recognize alpha-deoxyadenosine suggests that endonuclease IV is able to recognize a new class of DNA base lesions that is not recognized by other DNA N-glycosylases and AP endonucleases.
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PMID:Alpha-deoxyadenosine, a major anoxic radiolysis product of adenine in DNA, is a substrate for Escherichia coli endonuclease IV. 751 7

The objectives of this study were to elucidate the structural organization of the gene for human deoxyribonuclease I (DNase I) and to identify the mutation site underlying its classical genetic polymorphism. In order to determine the organization of this gene, we utilized a combination of direct polymerase chain reaction (PCR)-amplification of human genomic DNA and isolation of the overlapping clones from a cosmid human genomic library. Restriction endonuclease mapping, Southern blotting and DNA sequencing showed that the DNase I gene was approximately 3.2 kilobases long, it comprised 9 (I-IX) exons separated by eight introns and its complete sequence was determined. The first exon contained only the non-translated sequences of mRNA. In addition to several putative regulatory elements, TATA-like and CAAT-like sequences were observed in the region upstream of the translation initiation codon. These results provide information that will help to understand the expression and regulation of DNase I. The isoelectric focusing patterns of human DNase I showed that it exhibits classical genetic polymorphism (Kishi et al. 1989, 1990). A comparison of the entire translated sequences of the DNase I gene from two pairs of individuals with common DNase I phenotypes 1 and 2 revealed only one nucleotide residue difference in exon VIII, A for phenotype 1 and G for phenotype 2, thus producing Gln and Arg amino acid substitutions respectively at position 222 from the NH2-terminus of the mature enzyme. The predicted charge changes attributable to these amino acid substitutions are entirely consistent with the isoelectric focusing profiles of these two DNase I isozymes. We conclude that this substitution is solely responsible for the classical polymorphism of DNase I protein.
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PMID:Structure of the human deoxyribonuclease I (DNase I) gene: identification of the nucleotide substitution that generates its classical genetic polymorphism. 776 78

A genomic DNA obtained from a female patient with complete b-subunit deficiency was examined by Southern blotting analysis and in vitro amplification. Nucleotide sequence analysis showed that adenosine-4161 at the acceptor splice junction of intron A/exon II was deleted in half of the amplified DNAs, resulting in a loss of the obligatory AG splicing sequence. The absence of adenosine-4161 was confirmed by cleavage with TaqI endonuclease of the amplified DNAs. Moreover, sequence analysis showed that guanosine-11499 coding for Cys 430 (TGC) in exon VIII was replaced by thymidine in half of the amplified DNAs, resulting in an amino acid change to Phe (TTC) and the destruction of a disulfide bond in the seventh Sushi domain. This mutation was also confirmed by cleavage with MboII endonuclease. Thus, the proband turned out to be a compound heterozygote of two separate defective alleles. Although half of the amplified DNAs for exon VIII of her daughter or son were cleaved by MboII, those for intron A were not cleaved by TaqI. The replacement of guanosine-11499 by thymidine in their exon VIII has also been confirmed by sequence analysis, indicating that they are heterozygous for one normal and one defective allele.
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PMID:Two genetic defects in a patient with complete deficiency of the b-subunit for coagulation factor XIII. 832 18

Cholera is endemic in Bangladesh, and a regular seasonal pattern of cholera epidemics occurs. We examined the clonal relationships among 103 clinical and environmental Vibrio cholerae isolates belonging to O1, O139, or non-O1 non-O139 serogroups isolated during epidemic and interepidemic periods in Bangladesh and compared them with those of 51 V. cholerae isolates from four countries in Asia and Africa. These studies were done by a computer-assisted numerical analysis of the restriction endonuclease cleavage patterns of rRNA genes (ribotypes). Unweighed pair-group cluster analysis of BglI- and HindIII-generated band patterns revealed 16 clusters. Ribotypes were defined as clusters of strains possessing > 98% similarity. The results showed that 154 isolates could be differentiated into 15 different ribotypes, and strains belonging to 3 of these ribotypes (ribotypes I, V, and VIIIA and VIIIB) were isolated more frequently during the epidemic periods than during interepidemic periods in Bangladesh. Classical vibrios belonged to six different ribotypes (ribotypes I to VI), with a mean similarity coefficient of 0.84, and the El Tor vibrios belonged to five different ribotypes (ribotypes VIIIA and IX to XII), with a mean similarity coefficient of 0.82. A single clone of El Tor vibrios (ribotype XII) was resident in Tanzania, whereas Nigeria, Syria, and India shared toxigenic El Tor strains with Bangladesh. Cholera toxin (CT)-positive O139 vibrios isolated from Bangladesh and India belonged to a single ribotype (ribotype VIIIB) and were > 98% similar to one of the ribotypes of El Tor vibrios (ribotype VIIIA), but a CT-negative O139 vibrio from Argentina (ribotype XIII) was < 75% similar to the same cluster of El Tor vibrios, thus suggesting more than one possible origin for O139 vibrios. Strains belonging to the same ribotypes (ribotypes VIII to X) were isolated from both patients and surface water in Bangladesh, indicating possible transmission through surface water. A clone of a CT-positive environmental isolate of non-O1 V. cholerae (ribotype VII) was found to be closely related (76.3% similarity) to a clone of classical vibrios (ribotype I) and was only between 27.2 and 56.1% similar to clusters of El Tor, O139, and two other non-O1 nontoxigenic clones.
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PMID:Molecular epidemiology of toxigenic Vibrio cholerae in Bangladesh studied by numerical analysis of rRNA gene restriction patterns. 857 28

Recent investigations in our laboratory revealed divalent cation-dependent endonuclease activity in testes from 2-methoxyethanol-treated rats, which was able to cleave substrate DNA into a pattern of DNA fragmentation consisting of approximately 180-200 base pairs. Further studies were undertaken to characterize the active nuclease. F344 rats were treated with 2-methoxyethanol, a glycol ether that causes the death of pachytene spermatocytes in juvenile and adult rats. The active nuclease was found in nuclear extract from treated animals, but not controls. A radioactive gel nuclease assay, which detects degradation and loss of 32P-labeled DNA from a DNA-containing polyacrylamide gel, localized the nuclease activity to a band of approximately 18 kDa. This activity was dependent on calcium and was inhibited by both zinc and aurintricarboxylic acid. Amino acid sequence data showed that this protein was identical to cyclophilin A. Immunohistochemistry using antibodies against cyclophilin A found specific staining in pachytene spermatocytes, spermatids, interstitial cells, and Sertoli cell nuclei. Cyclophilin A staining was present in both control and 2-methoxyethanol-treated rat testes in a stage-dependent manner, with pachytene spermatocytes in stage-VIII-XIV seminiferous tubules most heavily stained. These data demonstrate that rat testis germ cells contain relatively high levels of cyclophilin A whose nuclease activity is associated with spermatocyte apoptosis induced by 2-methoxyethanol.
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PMID:Cyclophilin A is present in rat germ cells and is associated with spermatocyte apoptosis. Reproductive Toxicology Group. 911 44

BssHII restriction endonuclease cleaves 5'-GCGCGC-3' on double-stranded DNA between the first and second bases to generate a four base 5'overhang. BssHII restriction endonuclease was purified from the native Bacillus stearothermophilus H3 cells and its N-terminal amino acid sequence was determined. Degenerate PCR primers were used to amplify the first 20 codons of the BssHII restriction endonuclease gene. The BssHII restriction endonuclease gene (bssHIIR) and the cognate BssHII methyltransferase gene (bssHIIM) were cloned in Escherichia coli by amplification of Bacillus stearothermophilus genomic DNA using PCR and inverse PCR. BssHII methyltransferase (M.BssHII) contains all 10 conserved cytosine-5 methyltransferase motifs, but motifs IX and X precede motifs I-VIII. Thus, the conserved motifs of M. BssHII are circularly permuted relative to the motif organizations of other cytosine-5 methyltransferases. M.BssHII and the non-cognate multi-specific phiBssHII methyltransferase, M.phiBss HII [Schumann,J. et al . (1995) Gene, 157, 103-104] share 34% identity in amino acid sequences from motifs I-VIII, and 40% identity in motifs IX-X. A conserved arginine is located upstream of a TV dipeptide in the N-terminus of M.BssHII that may be responsible for the recognition of the guanine 5' of the target cytosine. The BssHII restriction endonuclease gene was expressed in E.coli via a T7 expression vector.
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PMID:Cloning of the BssHII restriction-modification system in Escherichia coli : BssHII methyltransferase contains circularly permuted cytosine-5 methyltransferase motifs. 932 48

Escherichia coli endonuclease VIII (endo VIII) was identified as an enzyme that, like endonuclease III (endo III), removes radiolysis products of thymine including thymine glycol, dihydrothymine, beta-ureidoisobutyric acid, and urea from double-stranded plasmid or phage DNA and cleaves the DNA strand at abasic (AP) sites (Melamede, R. J., Hatahet, Z., Kow, Y. W., Ide., H., and Wallace, S. S. (1994) Biochemistry 33, 1255-1264). Using apparently homogeneous endo VIII protein, we now show that endo VIII removes from double-stranded oligodeoxyribonucleotides the stable oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil. Endo VIII cleaved the damage-containing DNA strand by beta,delta-elimination as does formamidopyrimidine DNA glycosylase (Fpg). Like Fpg, endo VIII also excised the 5'-terminal deoxyribose phosphate from an endonuclease IV (endo IV) pre-incised AP site. Thus, in addition to amino acid sequence homology (Jiang, D., Hatahet, Z., Blaisdell, J., Melamede, R. J., and Wallace, S. S. (1997) J. Bacteriol. 179, 3773-3782), endo VIII shares a number of catalytic properties with Fpg. In addition, endo VIII specifically bound to oligodeoxynucleotides containing a reduced AP site with a stoichiometry of 1:1 for protein to DNA with an apparent equilibrium dissociation constant of 3.9 nM. Like Fpg and endo III, the DNase I footprint was small with contact sites primarily on the damage-containing strand; unlike Fpg and endo III, the DNA binding of endo VIII to DNA was asymmetric, 3' to the reduced AP site.
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PMID:Characterization of Escherichia coli endonuclease VIII. 940 26

Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5' or 3' of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5' or 3' was reduced compared with cleavage at positions three or six 5' or 3'. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5' or 3' in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3' of the lesion than when the gap was 3 nt 3' of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.
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PMID:Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII. 946 50

The Bpu 10I R-M system from Bacillus pumilus 10, which recognizes the asymmetric 5'-CCTNAGC sequence, has been cloned, sequenced and expressed in Escherichia coli . The system comprises four adjacent, similarly oriented genes encoding two m5C MTases and two subunits of Bpu 10I ENase (34.5 and 34 kDa). Both bpu10IR genes either in cis or trans are needed for the manifestation of R. Bpu 10I activity. Subunits of R. Bpu 10I, purified to apparent homogeneity, are both required for cleavage activity. This heterosubunit structure distinguishes the Bpu 10I restriction endonuclease from all other type II restriction enzymes described previously. The subunits reveal 25% amino acid identity. Significant similarity was also identified between a 43 amino acid region of R. Dde I and one of the regions of higher identity shared between the Bpu 10I subunits, a region that could possibly include the catalytic/Mg2+binding center. The similarity between Bpu 10I and Dde I MTases is not limited to the conserved motifs (CM) typical for m5C MTases. It extends into the variable region that lies between CMs VIII and IX. Duplication of a progenitor gene, encoding an enzyme recognizing a symmetric nucleotide sequence, followed by concerted divergent evolution, may provide a possible scenario leading to the emergence of the Bpu 10I ENase, which recognizes an overall asymmetric sequence and cleaves within it symmetrically.
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PMID:Cloning and analysis of the four genes coding for Bpu10I restriction-modification enzymes. 946 72


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