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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)

A mutant allele of the Escherichia coli nfo gene encoding endonuclease IV, nfo-186, was cloned into plasmid pUC18. When introduced into an E. coli xthA nfo mutant, the gene product of nfo-186 complemented the hypersensitivity of the mutant to methyl methanesulfonate (MMS) but not to hydrogen peroxide (H2O2) and bleomycin. These results suggest that the mutant endonuclease IV has normal activity for repairing DNA damages induced by MMS but not those induced by H2O2 and bleomycin. A missense mutation in the cloned nfo-186 gene, in which the wild-type glycine 149 was replaced by aspartic acid, was detected by DNA sequencing. The wild-type and mutant endonuclease IV were purified to near homogeneity, and their apurinic (AP) endonuclease and 3'-phosphatase activities were determined. No difference was observed in the AP endonuclease activities of the wild-type and mutant proteins. However, 3'-phosphatase activity was dramatically reduced in the mutant protein. From these results, it is concluded that the endonuclease IV186 protein is specifically deficient in the ability to remove 3'-terminus-blocking damage, which is required for DNA repair synthesis, and it is possible that the lethal DNA damage by H2O2 is 3'-blocking damage and not AP-site damage.
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PMID:A mutant endonuclease IV of Escherichia coli loses the ability to repair lethal DNA damage induced by hydrogen peroxide but not that induced by methyl methanesulfonate. 128 Feb 56

An endonuclease with 3'-nucleotidase activity (nuclease Le1) was purified from fruit bodies of Lentinus edodes in a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The apparent molecular weight of nuclease Le1 was about 27000. The nuclease was inactivated in the presence of ethylenediaminetetraacetic acid (EDTA) and reactivated by the addition of Zn2+. Hydrolysis of poly U by the nuclease showed many intermediate size oligomers prior to the formation of 5'-uridine monophosphate (UMP). Therefore, it was concluded that nuclease Le1 was a Zn(2+)-endonuclease similar to P1-nuclease from Penicillium citrinum. The nuclease was very sensitive to ionic strength, but pH-profiles of the hydrolysis of four 3'-nucleotides were very similar to those of P1 nuclease from P. citrinum.
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PMID:Purification and characterization of a nuclease from Lentinus edodes. 172 78

We have developed a strategy by which the nature of phosphodiester bond breaks produced by various DNA-repair endonucleases and also other nucleases, can be characterized. A purified apurinic/apyrimidinic (AP) specific endonuclease from a permanently established mouse plasmacytoma cell-line (MPC-11) has been examined with respect to the exact incision site generated at the baseless site. By the aid of enzymatic treatment with calf intestinal phosphatase, the 3'-phosphatase activity of T4-polynucleotide kinase, chemical modification with piperidine in addition to the Maxam-Gilbert sequencing procedure, followed by separation on a DNA-sequencing gel, the nature of the cleaved phosphodiester bond, both 3' and 5' to the cleavage site, has been established. The AP-specific endonuclease investigated was classified as a class II AP-endonuclease according to the four possible classes of AP-endonuclease with respect to the termini produced. By use of this technique each single damaged and cleaved site can be investigated separately.
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PMID:Analysis of cleavage products of DNA repair enzymes and other nucleases. Characterization of an apurinic/apyrimidinic specific endonuclease from mouse plasmacytoma cells. 245 3

Histones and polyamines nick the phosphodiester bond 3' to AP (apurinic/apyrimidinic) sites in DNA by inducing a beta-elimination reaction, which can be followed by delta-elimination. These beta- and delta-elimination reactions might be important for the repair of AP sites in chromatin DNA in either of two ways. In one pathway, after the phosphodiester bond 5' to the AP site has been hydrolysed with an AP endonuclease, the 5'-terminal base-free sugar 5'-phosphate is released by beta-elimination. The one-nucleotide gap limited by 3'-OH and 5'-phosphate ends is then closed by DNA polymerase-beta and DNA ligase. We have shown in vitro that such a repair is possible. In the other pathway, the nicking 3' to the AP site by beta-elimination occurs first. We have shown that the 3'-terminal base-free sugar so produced cannot be released by the chromatin AP endonuclease from rat liver. But it can be released by delta-elimination, leaving a gap limited by 3'-phosphate and 5'-phosphate. After conversion of the 3'-phosphate into a 3'-OH group by the chromatin 3'-phosphatase, there will be the same one-nucleotide gap, limited by 3'-OH and 5'-phosphate, as that formed by the successive actions of the AP endonuclease and the beta-elimination catalyst in the first pathway.
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PMID:Possible roles of beta-elimination and delta-elimination reactions in the repair of DNA containing AP (apurinic/apyrimidinic) sites in mammalian cells. 246 81

Escherichia coli endonuclease IV hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free deoxyribose. It also hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free 2',3'-unsaturated sugar produced by nicking 3' to an AP (apurinic or apyrimidinic) site by beta-elimination; this explains why the unproductive end produced by beta-elimination is converted by the enzyme into a 3'-OH end able to prime DNA synthesis. The action of E. coli endonuclease IV on an internal AP site is more complex: in a first step the C(3')-O-P bond 5' to the AP site is hydrolysed, but in a second step the 5'-terminal base-free deoxyribose 5'-phosphate is lost. This loss is due to a spontaneous beta-elimination reaction in which the enzyme plays no role. The extreme lability of the C(3')-O-P bond 3' to a 5'-terminal AP site contrasts with the relative stability of the same bond 3' to an internal AP site; in the absence of beta-elimination catalysts, at 37 degrees C the half-life of the former is about 2 h and that of the latter 200 h. The extreme lability of a 5'-terminal AP site means that, after nicking 5' to an AP site with an AP endonuclease, in principle no 5'----3' exonuclease is needed to excise the AP site: it falls off spontaneously. We have repaired DNA containing AP sites with an AP endonuclease (E. coli endonuclease IV or the chromatin AP endonuclease from rat liver), a DNA polymerase devoid of 5'----3' exonuclease activity (Klenow polymerase or rat liver DNA polymerase beta) and a DNA ligase. Catalysts of beta-elimination, such as spermine, can drastically shorten the already brief half-life of a 5'-terminal AP site; it is what very probably happens in the chromatin of eukaryotic cells. E. coli endonuclease IV also probably participates in the repair of strand breaks produced by ionizing radiations: as E. coli endonuclease VI/exonuclease III, it is a 3'-phosphoglycollatase and also a 3'-phosphatase. The 3'-phosphatase activity of E. coli endonuclease VI/exonuclease III and E. coli endonuclease IV can also be useful when the AP site has been excised by a beta delta-elimination reaction.
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PMID:The multiple activities of Escherichia coli endonuclease IV and the extreme lability of 5'-terminal base-free deoxyribose 5-phosphates. 247 13

The amino acid composition and NH2-terminal amino acid sequence of barley nuclease (EC 3.1.30.2) were determined. The amino acid composition is similar to that of mung bean nuclease, and therefore the biochemical properties of barley nuclease were characterized and compared with those of mung bean and other plant nucleases. The 3'-nucleotidase activity of barley nuclease is greater for purine than for pyrimidine ribonucleotides. The enzyme has little activity towards ribonucleoside 2' and 5'-monophosphates, and deoxyribonucleoside 3' and 5'-monophosphates, and is also inactive towards the 3'-phosphoester linkage of nucleoside cyclic 2',3' and 3',5'-monophosphates. The enzyme hydrolyzes dinucleoside monophosphates, showing strong preference for purine nucleosides as the 5' residues. Barley nuclease shows significant base preference for homoribonucleic acids, catalyzing the hydrolysis of polycytidylic acid greater than polyuridylic acid greater than polyadenylic acid much greater than polyguanylic acid. The enzyme also has preference for single-stranded nucleic acids. Hydrolysis of nucleic acids is primarily endonucleolytic, whereas the products of digestion possess 5'-phosphomonoester groups. Nuclease activity is inhibited by ethylenediaminetetraacetic acid and zinc is required for reactivation. Secretion of nuclease from barley aleurone layers is dependent on the hormone gibberellic acid [Brown, P.H. and Ho, T.-h. D. (1986) Plant Physiol. 82, 801-806]. Consistent with these results, gibberellic acid induces up to an eight-fold increase in the de novo synthesis of nuclease in aleurone layers. The secreted enzyme is a glycoprotein having an apparent molecular mass of 35 kDa. It consists of a single polypeptide having an asparagine-linked, high-mannose oligosaccharide. The protein portion of the molecule has a molecular mass of 33 kDa.
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PMID:Biochemical properties and hormonal regulation of barley nuclease. 282 11

We have characterized a 3'-nucleotidase activity of T. brucei. The enzyme has a pH optimum of 8.7, is inactivated by chelating agents and stimulated by divalent cations. It is inhibited by Zn2+, Mn2+, pyrophosphate and the trypanocidal drug suramin for which it has a Ki of 3 microM. From cell fractionation experiments it is concluded that the enzyme is located in the plasma membrane. Alkaline 3'-endoribonuclease is also located in the plasma membrane of T. brucei and this activity shares a great number of properties with the 3'-nucleotidase activity, including its sensitivity to suramin. The possibility that both 3'-nucleotidase and endonuclease activities are catalyzed by the same enzyme cannot be excluded.
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PMID:Localization of 3'-nucleotidase and calcium-dependent endoribonuclease in the plasma-membrane of Trypanosoma brucei. 288 57

Drosophila Rrp1 has several tightly associated enzymatic activities, including double-strand DNA 3'-exonuclease, apurinic/apyrimidinic endonuclease, 3'-phosphatase, and 3'-phosphodiesterase. The carboxyl-terminal third of Rrp1, homologous to Escherichia coli exonuclease III, is sufficient to repair oxidative and alkylation-induced DNA damage in vivo. Using a screen for partial complementation of repair-deficient E. coli, we isolated three mutants of the nuclease domain of Rrp1: T462A, K463Q, and L484P, that protect against methyl methanesulfonate (MMS)-induced but not t-BuO2H-induced DNA damage. Thr-462 and Lys-463 are highly conserved residues found in a cluster of 5 conserved amino acids (LQETK), while Leu-484 is poorly conserved. Gln-460 Glu-461, Thr-462, and Lys-463 and Leu-484 were altered by site-directed mutagenesis using a plasmid including the entire Rrp1 gene and mutant proteins were purified. Mutants of the three residues Glu-461, Thr-462, and Lys-463 demonstrate 8-200-fold lower phosphodiesterase specific activity than wild-type Rrp1. E461A has a 30-fold reduction in AP endonuclease and is MMS-sensitive, but all other mutants have near-normal AP endonuclease and are MMS-resistant. Glu-461 appears to be essential for the nuclease function for Rrp1. Lys-463 and, to a lesser extent, Thr-462 influence the substrate specificity of the Rrp1 nuclease.
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PMID:Single amino acid changes alter the repair specificity of Drosophila Rrp1. Isolation of mutants deficient in repair of oxidative DNA damage. 779 76

Drosophila Rrp1 (recombination repair protein 1) is a DNA repair enzyme whose nuclease activities include AP-endonuclease, 3'-exonuclease, 3'-phosphodiesterase and 3'-phosphatase. This study investigates the sequence specificity of the dsDNA 3'-exonuclease activity of Rrp1. We demonstrate that the activity is more efficient in purine-rich regions of dsDNA than in pyrimidine-rich regions. Rrp1 exonuclease activity is examined at 3'-terminal homopurine or homopyrimidine tracts, at junctions between purine- and pyrimidine-rich sequences and upon encountering repeated dinucleotide runs. The data show that purine-purine and 3'-pyrimidine-5'-purine dinucleotide bonds are cleaved faster than 3'-purine-5'-pyrimidine or pyrimidine-pyrimidine bonds. Thus, the base occupying the penultimate position in the 3'-terminal dinucleotide may be important in determining the relative efficiency of bond cleavage by Rrp1. These findings may reflect upon specific DNA-protein interactions in the enzyme active site.
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PMID:Drosophila Rrp1 3'-exonuclease: demonstration of DNA sequence dependence and DNA strand specificity. 891 93

Tpp1 is a DNA 3'-phosphatase in Saccharomyces cerevisiae that is believed to act during strand break repair. It is homologous to one domain of mammalian polynucleotide kinase/3'-phosphatase. Unlike in yeast, we found that Tpp1 could confer resistance to methylmethane sulfonate when expressed in bacteria that lack abasic endonuclease/3'-phosphodiesterase function. This species difference was due to the absence of delta-lyase activity in S. cerevisiae, since expression of bacterial Fpg conferred Tpp1-dependent resistance to methylmethane sulfonate in yeast lacking the abasic endonucleases Apn1 and Apn2. In contrast, beta-only lyases increased methylmethane sulfonate sensitivity independently of Tpp1, which was explained by the inability of Tpp1 to cleave 3' alpha,beta-unsaturated aldehydes. In parallel experiments, mutations of TPP1 and RAD1, encoding part of the Rad1/Rad10 3'-flap endonuclease, caused synthetic growth defects in yeast strains lacking Apn1. In contrast, Fpg expression led to a partial rescue of apn1 apn2 rad1 synthetic lethality by converting lesions into Tpp1-cleavable 3'-phosphates. The collected experiments reveal a profound toxicity of strand breaks with irreparable 3' blocking lesions, and extend the function of the Rad1/Rad10 salvage pathway to 3'-phosphates. They further demonstrate a role for Tpp1 in repairing endogenously created 3'-phosphates. The source of these phosphates remains enigmatic, however, because apn1 tpp1 rad1 slow growth could be correlated with neither the presence of a yeast delta-lyase, the activity of the 3'-phosphate-generating enzyme Tdp1, nor levels of endogenous oxidation.
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PMID:The role of yeast DNA 3'-phosphatase Tpp1 and rad1/Rad10 endonuclease in processing spontaneous and induced base lesions. 1278 66


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