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)

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

An enzyme that plays an important role in the repair of oxidative DNA damage is the 3'-phosphodiesterase. This activity, which repairs damaged DNA 3'-termini,can be detected using several available biochemical assays. We present a method to detect 3'-phosphodiesterase activity of renatured proteins immobilized in polyacrylamide gels. The model substrate, labeled with [alpha-32P]dCTP, contains 3'-phosphoglycolate termini produced by bleomycin-catalyzed cleavage of the self-complementary alternating copolymer poly(dGdC). The DNA substrate is incorporated into the gel matrix during standard SDS-PAGE. Active 3'-phosphodiesterase enzymes are detected visibly by the loss of radioactivity at a position corresponding to the mobility of the enzyme during SDS-PAGE. Using this procedure, two Escherichia coli 3'-phosphodiesterases, exonuclease III and endonuclease IV, are readily detected in crude cell extracts or as homogeneous purified proteins. Extracts of mutant cells lack activity at the positions of exonuclease III and endonuclease IV but retain activity in the position of a much larger protein (Mr approximately 100 kDa). The identification of this novel 100 kDa E.coli 3'-phosphodiesterase demonstrates the potential value of the activity gel method described here.
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PMID:In situ activity gel for DNA repair 3'-phosphodiesterase. 910 75

Earlier work indicates that the major DNA repair phosphodiesterase (PDE) in yeast cells is the well-characterized Apn1 protein. Apn1 demonstrates both Mg2+-independent PDE activity and Mg2+-independent class II apurinic/apyrimidinic (AP) endonuclease activity and represents greater than 90% of the activity detected in crude extracts from wild-type yeast cells. Apn1 is related to Echerichia coli endonuclease IV, both in its enzymatic properties and its amino acid sequence. In this work, we report the partial purification of a novel yeast protein, Pde1, present in Apn1-deficient cells. Pde1 is purified by sequential BioRex-70, PBE118, and MonoS chromatography steps using a sensitive and highly specific 3'-phosphoglycolate-terminated oligonucleotide-based assay as a measure of PDE activity. Mg2+-stimulated PDE and Mg2+-stimulated class II AP endonuclease copurify during this procedure. These results indicate that yeast, like many other organisms studied to date, has enzymatic redundancy for the repair of 3'-blocking groups and abasic sites.
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PMID:Partial purification of Pde1 from Saccharomyces cerevisiae: enzymatic redundancy for the repair of 3'-terminal DNA lesions and abasic sites in yeast. 916 80

We describe here a sensitive and straightforward method for characterizing the methylation specificity of type II DNA methyltransferase (MTase) using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. DNA substrate, prepared by ligation of a commercially available oligonucleotide, was modified by the subject MTase, and was derivatized to a mixture of single-stranded oligonucleotides through endonuclease treatment, heat-denaturation and limited digestion by 3'-terminus-specific phosphodiesterase I. MALDI-TOF mass spectrometry was used to determine the mass differences between the digestion products, and the methylated nucleotide was explicitly identified by the mass increase of 14 Da due to the base modification. The method was applicable to the three representative MTases M. Eco RI, M. Bam HI and M. Hae III.
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PMID:Determination of methylation specificity of sequence-specific DNA methyltransferases using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. 932 74

Uracil can arise in DNA by misincorporation of dUTP into nascent DNA and/or by cytosine deamination in established DNA. Based on recent findings, both pathways appear to be promoted in the methyl-deficient model of hepatocarcinogenesis. A chronic increase in the ratio dUTP:dTTP with folate/methyl deficiency can result in a futile cycle of excision and reiterative uracil misincorporation leading to premutagenic apyrimidinic (AP) sites, DNA strand breaks, DNA fragmentation and apoptotic cell death. The progressive accumulation of unmethylated cytosines with chronic methyl deficiency will increase the potential for cytosine deamination to uracil and further stress uracil mismatch repair mechanisms. Uracil is removed by a highly specific uracil-DNA glycosylase (UDG) leaving an AP site that is subsequently repaired by sequential action of AP endonuclease, 5'-phosphodiesterase, a DNA polymerase and DNA ligase. Since the DNA polymerases cannot distinguish between dUTP and dTTP, an increase in dUTP:dTTP ratio will promote uracil misincorporation during both DNA replication and repair synthesis. The misincorporation of uracil for thymine (5-methyluracil) may constitute a genetically significant form of DNA hypomethylation distinct from cytosine hypomethylation. In the present study a significant increase in the level of uracil in liver DNA as early as 3 weeks after initiation of folate/methyl deficiency was accompanied by parallel increases in DNA strand breaks, AP sites and increased levels of AP endonuclease mRNA. In addition, uracil was also detected within the p53 gene sequence using UDG PCR techniques. Increased levels of uracil in DNA implies that the capacity for uracil base excision repair is exceeded with chronic folate/methyl deficiency. It is possible that enzyme-induced extrahelical bases, AP sites and DNA strand breaks interact to negatively affect the stability of the DNA helix and stress the structural limits of permissible uracil base excision repair activity. Thus substitution of uracil for thymine induces repair-related premutagenic lesions and a novel form of DNA hypomethylation that may relate to tumor promotion in the methyl-deficient model of hepatocarcinogenesis.
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PMID:Presence and consequence of uracil in preneoplastic DNA from folate/methyl-deficient rats. 939 4

Using complementation tests and nucleotide sequencing, we showed that the rad58-4 mutation was an allele of the MRE11 gene and have renamed the mutation mre11-58. Two amino acid changes from the wild-type sequence were identified; one is located at a conserved site of a phosphodiesterase motif, and the other is a homologous amino acid change at a nonconserved site. Unlike mre11 null mutations, the mre11-58 mutation allowed meiosis-specific double-strand DNA breaks (DSBs) to form at recombination hot spots but failed to process those breaks. DSB ends of this mutant were resistant to lambda exonuclease treatment. These phenotypes are similar to those of rad50S mutants. In contrast to rad50S, however, mre11-58 was highly sensitive to methyl methanesulfonate treatment. DSB end processing induced by HO endonuclease was suppressed in both mre11-58 and the mre11 disruption mutant. We constructed a new mre11 mutant that contains only the phosphodiesterase motif mutation of the Mre11-58 protein and named it mre11-58S. This mutant showed the same phenotypes observed in mre11-58, suggesting that the phosphodiesterase consensus sequence is important for nucleolytic processing of DSB ends during both mitosis and meiosis.
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PMID:A novel mre11 mutation impairs processing of double-strand breaks of DNA during both mitosis and meiosis. 941 73

The pseudorabies virus (PRV) DNase gene has previously been mapped within the PRV genome. To characterize further the enzymic properties of PRV DNase, this enzyme was expressed in Escherichia coli with the use of a pET expression vector. The protein was purified to homogeneity and assayed for nuclease activity in vitro. Recombinant PRV DNase exhibited an alkaline pH preference and an absolute requirement for Mg2+ ions that could not be replaced by Ca2+ and Na+ ions. Further studies showed that PRV DNase exhibited endonuclease, 5'-exonuclease and 3'-exonuclease activities in both single-stranded and double-stranded DNA. This activity occurred randomly and no significant base preference was demonstrated. The multiple biochemical activities of PRV DNase are similar to the activities of Neurospora crassa endo-exonuclease and E. coli RecBCD, two additional enzymes that are involved in recombination. Taken together, the similarity of action between N. crassa endo-exonuclease, E. coli RecBCD, and PRV DNase suggests that PRV DNase might have a role in the process of recombination that occurs during PRV infection.
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PMID:Recombinant pseudorabies virus DNase exhibits a RecBCD-like catalytic function. 946 90

The bacteria Escherichia coli contains several exonucleases acting on both double- and single-stranded DNA and in both a 5'-->3' and 3'-->5' direction. These enzymes are involved in replicative, repair and recombination functions. We have identified a new exonuclease found in E.coli, termed exonuclease IX, that acts preferentially on single-stranded DNA as a 3'-->5' exonuclease and also functions as a 3'-phosphodiesterase on DNA containing 3'-incised apurinic/apyrimidinic (AP) sites to remove the product trans -4-hydroxy-2-pentenal 5-phosphate. The enzyme showed essentially no activity as a deoxyribophosphodiesterase acting on 5'-incised AP sites. The activity was isolated as a glutathione S-transferase fusion protein from a sequence of the E.coli genome that was 60% identical to a 260 bp region of the small fragment of the DNA polymerase I gene. The protein has a molecular weight of 28 kDa and is free of AP endonuclease and phosphatase activities. Exonuclease IX is expressed in E.coli , as demonstrated by reverse transcription-PCR, and it may function in the DNA base excision repair and other pathways.
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PMID:Exonuclease IX of Escherichia coli. 959 42

We describe physicochemical and enzymatic properties of 5' bridging phosphorothioester linkages at specific sites in DNA oligonucleotides. The susceptibility to hydrolysis at various pH values is examined and no measurable hydrolysis is observed at pH 5-9 after 4 days at 25 degrees C. The abilities of three 3'- and 5'-exonuclease enzymes to hydrolyze the DNA past this linkage are examined and it is found that the linkage causes significant pauses at the sulfur linkage for T4 DNA polymerase and calf spleen phosphodiesterase, but not for snake venom phosphodiesterase. Restriction endonuclease (Nsi I) cleavage is also attempted at a 5'-thioester junction and strong resistance to cleavage is observed. Also tested is the ability of polymerase enzymes to utilize templates containing single 5'-S-thioester linkages; both Klenow DNA polymerase and T7 RNA polymerase are found to synthesize complementary strands successfully without any apparent pause at the sulfur linkage. Finally, the thermal stabilities of duplexes containing such linkages are measured; results show that T m values are lowered by a small amount (2 degrees C) when one or two thioester linkages are present in an otherwise unmodified duplex. The chemical stability and surprisingly small perturbation by the 5' bridging sulfur make it a good candidate as a physical and mechanistic probe for specific protein or metal interactions involving this position in DNA.
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PMID:Chemical and enzymatic properties of bridging 5'-S-phosphorothioester linkages in DNA. 962 13

The flap endonuclease gene homologue from the hyperthermophilic archaeon, Pyrococcus horikoshii, was overexpressed in Escherichia coli and purified. The results of gel filtration indicated that this protein was a 41-kDa monomer. P. horikoshii flap endonuclease (phFEN) cleaves replication fork-like substrates (RF) and 5' double-strand flap structures (DF) using both flap endonuclease and 5'-3'-exonuclease activities. The mammalian flap endonuclease (mFEN) is a single-strand flap-specific endonuclease (Harrington, J. J., and Lieber, M. R. (1994) EMBO J. 13, 1235-1246), but the action patterns of phFEN appear to be quite different from those of mFEN at this point. The DF-specific flap endonuclease and 5'-exonuclease activities have not yet been reported. Therefore, this is the first report of the specific endo/exonuclease activities of phFEN. The DF-specific 5'-exonuclease activity degraded the downstream primer of 3' single-flap structure and was 15 times higher than the activities against nicked substrates without 3' flap strand. DF-specific flap endonuclease cleaved the 5' double-flap strand in DF and the lagging strand in RF at the junction portion. Because the RF appears to be the intermediate structure, due to the arrest of the replication fork, the double strand breaks after the arrests of the replication forks are probably caused by phFEN.
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PMID:Thermostable flap endonuclease from the archaeon, Pyrococcus horikoshii, cleaves the replication fork-like structure endo/exonucleolytically. 1037 33


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