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:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Repair of damaged DNA is of great importance in maintaining genome integrity, and there are several pathways for repair of damaged DNA in almost all organisms. Base excision repair (BER) is a main process for repairing DNA carrying slightly damaged bases. Several proteins are required for BER; these include DNA glycosylases, AP endonuclease, DNA polymerase, and DNA ligase. In some bacteria the single-stranded specific exonuclease, RecJ, is also involved in BER. In this research, six Chlamydiophila pneumoniae (C. pneumoniae) genes, encoding uracil DNA glycosylase (CpUDG), endonuclease IV (CpEndoIV), DNA polymerase I (CpDNApolI), endonuclease III (CpEndoIII), single-stranded specific exonuclease RecJ (CpRecJ), and DNA ligase (CpDNALig), were inserted into the expression vector pET28a. All proteins, except for CpDNALig, were successfully expressed in E. coli, and purified proteins were characterized in vitro. C. pneumoniae BER was reconstituted in vitro with CpUDG, CpEndoIV, CpDNApolI and E. coli DNA ligase (EcDNALig). After uracil removal by CpUDG, the AP site could be repaired by two BER pathways that involved in the replacement of either one (short patch BER) or multiple nucleotides (long patch BER) at the lesion site. CpEndoIII promoted short patch BER via its 5'-deoxyribophosphodiesterase (5'-dRPase) activity, while CpRecJ had little effect on short patch BER. The flap structure generated during DNA extension could be removed by the 5'-exonuclease activity of CpDNApolI. Based on these observations, we propose a probable mechanism for BER in C. pneumoniae.
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PMID:The mechanism of base excision repair in Chlamydiophila pneumoniae. 1608 68

We recently demonstrated that African swine fever virus DNA polymerase X (Pol X) is extremely error-prone during single-nucleotide gap-filling and that the downstream ASFV DNA ligase seals 3' mismatched nicks with high efficiency. To further assess the credence of our hypothesis that these proteins may promote viral diversification by functioning within the context of an aberrant DNA repair pathway, herein we characterize the third protein expected to function in this system, a putative AP endonuclease (APE). Assays of the purified protein using oligonucleotide substrates unequivocally establish canonical APE activity, 3'-phosphatase and 3'-phosphodiesterase activities (in the context of a single-nucleotide gap), 3' --> 5' exonuclease activity (in the context of a nick), and nucleotide incision repair activity against 5,6-dihydrothymine. The 3' --> 5' exonuclease activity is shown to be highly dependent upon the identity of the nascent 3' base pair and to be inhibited when 2-deoxyribose-5-phosphate, rather than phosphate, constitutes the 5' moiety of the nick. ASFV APE retains activity when assayed in the presence of EDTA but is inactivated by incubation with 1,10-phenanthroline in the absence of a substrate, suggesting that it is an endonuclease IV homologue possessing intrinsic metal cofactors. The activities of ASFV APE, when considered alongside those of Pol X and ASFV DNA ligase, provide an enhanced understanding of (i) the types of damage that are likely to be sustained by the viral genome and (ii) the mechanisms by which the minimalist ASFV DNA repair pathway, consisting of just these three proteins, contributes to the fitness of the virus.
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PMID:Contributions of an endonuclease IV homologue to DNA repair in the African swine fever virus. 1650 34

DNA diagnostic has been moving from expensive, low-throughput, multistep methods to inexpensive, higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays. In this method, we describe a universal molecular beacon (U-MB) as the fluorescent tracer in the real-time PCR technique. A 5'-universal template primer (5'-UT primer) has been designed with a tail in complementary to the loop and 5'-side arm sequence of U-MB at the 5'-end of forward target specific primer. As PCR cycles increase, a new DNA fragment with a 5'-UT primer tail is synthesized, which is used as the template for next PCR cycle. As the reverse primer extends to the 5'-UT primer tail, the U-MB hybridized is displaced and the fluorescence from the fluorophore of the U-MB is quenched, indicating that the allele-specific PCR is in progress. This tracing system combined with an allele-specific reverse primer and vent (exo-) DNA polymerase, a polymerase that lacks 3'- to 5'-exonuclease activity, was used for the detection of point mutations of base G in codon 259 (AGA) of exon 7 of p53 gene on a panel of breast cancer individuals.
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PMID:Universal molecular beacon-based tracer system for real-time polymerase chain reaction. 1710 84

Isonucleosides have been attracting a lot of attention in recent years due to the chemical and enzymatic stability and potential anticancer and antiviral activities. We have reported some of the isonucleosides which exhibited significant anticancer activity and found that the oligonucleotide incorporated with isonucleoside could increase the enzymatic stability against the degradation by phosphodiesterase. In this paper, we investigated the recognition of the isonucleoside triphosphates 1-6 by Taq, Vent(exo(-)), DeepVent(exo(-)), 9 degrees Nm, and Therminator DNA polymerases by a non-radioactivity method. We found that most of the isonucleoside triphosphates can be recognized by various DNA polymerase and act as terminators. Isonucleoside triphosphates 2 and 6 can be incorporated as substrates into the primer at 3' terminus to lengthen the chain dependent on a DNA template by Vent(exo(-)) and DeepVent(exo(-)) DNA polymerases.
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PMID:Synthesis and recognition of novel isonucleoside triphosphates by DNA polymerases. 1732 Apr 3

In Escherichia coli, exonuclease I (ExoI) is a monomeric processive 3'-5' exonuclease that degrades single-stranded DNA. The enzyme has been implicated as primarily being involved in repairing frameshift mutations. The structure of the enzyme has previously been determined in a hexagonal space group at 2.4 A resolution. Here, the structure of ExoI in complex with a nucleotide product, thymidine 5'-monophosphate, is described in an orthorhombic space group at 1.5 A resolution. This new high-resolution structure provides some insight into the interactions involved in binding a nucleotide product. The conserved active site contains a unique metal-binding position when compared with orthologous sites in the Klenow fragment, T4 DNA polymerase and dnaQ. This unique difference is proposed to be a consequence of the repositioning of an important histidine, His181, away from the active site.
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PMID:Structure of Escherichia coli exonuclease I in complex with thymidine 5'-monophosphate. 1821 21

DNA polymerase selectivity is crucial for the survival of any living species, yet varies significantly among different DNA polymerases. Errors within DNA polymerase-catalyzed DNA synthesis result from the insertion of noncanonical nucleotides and extension of misaligned DNA substrates. The substrate binding characteristics among DNA polymerases are believed to vary in properties such as shape and tightness of the binding pocket, which might account for the observed differences in fidelity. Here, we employed 4'-alkylated nucleotides and primer strands bearing 4'-alkylated nucleotides at the 3'-terminal position as steric probes to investigate differential active site properties of human DNA polymerase beta (Pol beta) and the 3'-->5'-exonuclease-deficient Klenow fragment of E. coli DNA polymerase I (KF(exo-)). Transient kinetic measurements indicate that both enzymes vary significantly in active site tightness at both positions. While small 4'-methyl and -ethyl modifications of the nucleoside triphosphate perturb Pol beta catalysis, extension of modified primer strands is only marginally affected. Just the opposite was observed for KF(exo-). Here, incorporation of the modified nucleotides is only slightly reduced, whereas size augmentation of the 3'-terminal nucleotide in the primer reduces the catalytic efficiency by more than 7000- and 260,000-fold, respectively. NMR studies support the notion that the observed effects derive from enzyme substrate interactions rather than inherent properties of the modified substrates. These findings are consistent with the observed differential capability of the investigated DNA polymerases in fidelity such as processing misaligned DNA substrates. The results presented provide direct evidence for the involvement of varied steric effects among different DNA polymerases on their fidelity.
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PMID:Opposed steric constraints in human DNA polymerase beta and E. coli DNA polymerase I. 1862 54

Prevailing conventional microbial detection methods depend largely on microbial cultivation in selective media that requires several days. Polymerase chain reaction (PCR)-based methods, including quantitative reverse transcription PCR, are also rapid and useful methods for identification of target microbes, although for practical use they still suffer from disadvantages such as contamination problems and cost. Here we demonstrate that RNA-primed rolling circle amplification (RPRCA) using phi29 DNA polymerase, a precircularized probe, and SYBR Green II achieved real-time detection of specific messenger RNA (mRNA) from living microbes. The precircularized DNA probe was prepared by intramolecular ligation using CircLigase and treated by exonuclease I to eliminate uncircularized oligonucleotide, thereby significantly reducing potential noise by nonspecific amplified DNA by-products that affect successive RPRCA. When in vitro transcribed green fluorescent protein (GFP) mRNA was used as a primer, RPRCA could specifically detect at least 1 fmol of this mRNA in the presence of a precircularized probe that had a sequence complementary to the 3' terminus of mRNA without reverse transcription. This method could also detect expressed GFP mRNA present in 10 ng of total RNA isolated from Escherichia coli without DNase treatment. These data suggest that RPRCA has the potential to be a direct, rapid, and convenient method for detecting microbial mRNA.
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PMID:Direct detection of green fluorescent protein messenger RNA expressed in Escherichia coli by rolling circle amplification. 2023 Jul 71

We present an optimized procedure for freeze-drying and storing reagents for multiplex PCR followed by genotyping using a tag-array minisequencing assay with four color fluorescence detection which is suitable for microfluidic assay formats. A test panel was established for five cancer mutations in three codons (175, 248 and 273) of the tumor protein gene (TP53) and for 13 common single nucleotide polymorphisms (SNPs) in the TP53 gene. The activity of DNA polymerase was preserved for six months of storage after freeze-drying, and the half-life of activities of exonuclease I and shrimp alkaline phosphatase were estimated to 55 and 200 days, respectively. We conducted a systematic genotyping comparison using freeze-dried and liquid reagents. The accuracy of successful genotyping was 99.1% using freeze-dried reagents compared to liquid reagents. As a proof of concept, the genotyping protocol was carried out with freeze-dried reagents stored in reaction chambers fabricated by micromilling in a cyclic olefin copolymer substrate. The results reported in this study are a key step towards the development of an integrated microfluidic device for point-of-care DNA-based diagnostics.
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PMID:Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices. 2066 55

Mutations in the genes of corrective 3' --> 5'-exonucleases as well as in DNA polymerases lead to decrease in DNA biosynthesis accuracy all over genome. This is accompanied by the increase in mutagenesis and carcinogenesis probabilities. In this work, the activities of 3' --> 5'-exonucleases and DNA polymerases were studied in the extracts from normal and cancer cells of rodents and humans, and we are the first to measure their integral ratios. As example, in cultivated dermal fibroblasts of an adult human, the value of the ratio of activities of 3' --> 5'-exonucleases to DNA polymerase activity (3'-exo/pol) surpassed several folds the such a value for HeLa cells. Similar picture was observed during the comparison of normal fibroblasts of rat embryos and transformed fibroblasts of Chinese hamster A238. Experiments with cell-free extracts of some organs from healthy rats of various ages have shown that normal proliferating cells demonstrate higher 3' --> 5'-exonuclease activity and higher values of 3'-exo/pol that quiescent cells. Comparison of these data suggests a violation of the function of corrective 3' --> 5'-exonucleases in abnormally growing cancer cells.
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PMID:[Ratio of 3' --> 5'-exonuclease and DNA-polymerase activities in normal and cancer cells of rodents and humans]. 2096 97

The N-glycosidic bond can be hydrolyzed spontaneously or by glycosylases during removal of damaged bases by the base excision repair pathway, leading to the formation of highly mutagenic apurinic/apyrimidinic (AP) sites. Organisms encode for evolutionarily conserved repair machinery, including specific AP endonucleases that cleave the DNA backbone 5' to the AP site to prime further DNA repair synthesis. We report on the DNA polymerase X from the bacterium Bacillus subtilis (PolX(Bs)) that, along with polymerization and 3'-5'-exonuclease activities, possesses an intrinsic AP-endonuclease activity. Both, AP-endonuclease and 3'-5'-exonuclease activities are genetically linked and governed by the same metal ligands located at the C-terminal polymerase and histidinol phosphatase domain of the polymerase. The different catalytic functions of PolX(Bs) enable it to perform recognition and incision at an AP site and further restoration (repair) of the original nucleotide in a standalone AP-endonuclease-independent way.
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PMID:Intrinsic apurinic/apyrimidinic (AP) endonuclease activity enables Bacillus subtilis DNA polymerase X to recognize, incise, and further repair abasic sites. 2097 32


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