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)

Saccharomyces cerevisiae mtDNA polymerase, isolated as a single 135-kDa recombinant polypeptide, showed high processivity and a capacity of use poly(dA).oligo(dT), poly(rA).oligo(dT), or primed bacteriophage M13 DNA as a template. In a primer extension assay, the enzyme exhibited an intrinsic 3'-5'-exonuclease activity. By optimizing the polymerization reaction conditions, apparent Km and Vmax values could be determined for the incorporation of dTTP, 2'-3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZTTP), 3'-fluoro-TTP, dCTP, 2'-3'-dideoxy-CTP, and didehydro(d4)CTP. The yeast mtDNA polymerase used ddTTP, 3'-fluoro-TTP, and ddCTP almost as efficiently as natural deoxynucleoside trisphosphates. Both 3'AZTTP and d4CTP were each significantly less efficient as substrates. Overall, the kinetic data with mtDNA polymerase were very similar to those of the recombinant human immunodeficiency virus reverse transcriptase control. Terminally incorporated AZTTP or ddTTP was not removed by the 3'-5' exonuclease activity of mtDNA polymerase. This may explain the inhibition of mtDNA replication observed in anti-human immunodeficiency virus treatment with dideoxynucleoside analogs for their effects of mtDNA polymerase could be of value in future rational drug design.
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PMID:Efficient incorporation of anti-HIV deoxynucleotides by recombinant yeast mitochondrial DNA polymerase. 764 50

DNA exonucleases are critical for DNA replication, repair, and recombination. In the bacterium Escherichia coli there are 14 DNA exonucleases including exonucleases I-IX (including the two DNA polymerase I exonucleases), RecJ exonuclease, SbcCD exonuclease, RNase T, and the exonuclease domains of DNA polymerase II and III. Here we report the discovery and characterization of a new E. coli exonuclease, exonuclease X. Exonuclease X is a member of a superfamily of proteins that have homology to the 3'-5' exonuclease proofreading subunit (DnaQ) of E. coli DNA polymerase III. We have engineered and purified a (His)(6)-exonuclease X fusion protein and characterized its activity. Exonuclease X is a potent distributive exonuclease, capable of degrading both single-stranded and duplex DNA with 3'-5' polarity. Its high affinity for single-strand DNA and its rapid catalytic rate are similar to the processive exonucleases RecJ and exonuclease I. Deletion of the exoX gene exacerbated the UV sensitivity of a strain lacking RecJ, exonuclease I, and exonuclease VII. When overexpressed, exonuclease X is capable of substituting for exonuclease I in UV repair. As we have proposed for the other single-strand DNA exonucleases, exonuclease X may facilitate recombinational repair by pre-synaptic and/or post-synaptic DNA degradation.
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PMID:Exonuclease X of Escherichia coli. A novel 3'-5' DNase and Dnaq superfamily member involved in DNA repair. 1051 96

Human DNA apurinic/apyrimidinic endonuclease 1 (APE1) is involved in the DNA base excision repair process. In addition to its AP (apurinic/apyrimidinic) endonucleolytic function, APE1 possesses 3' phosphodiesterase and 3'-5' exonuclease activities. The 3'-5' exonuclease activity is considered important in proofreading of DNA synthesis catalyzed by DNA polymerase beta. Here, we examine the removal of matched and mismatched dNMP from the 3' terminus of the 3'-recessed and nicked DNA by the APE1 activity using two different reaction buffers. To investigate whether the ability of APE1 to excise nucleotides from the 3' terminus depends on the thermal stability of the DNA duplex, we studied this characteristic of the DNAs that were used in the exonuclease assays in these two buffers. Our data confirm that APE1 removes mismatched nucleotides from the 3' terminus of DNA more efficiently than matched pairs. Both the efficiency of the 3'-5' exonuclease activity of APE1 and the thermal stability of DNA duplexes varied depending on the nature of the flanking group at the 5' margin of the nick. The 3'-5' exonuclease activity of APE1 shows a preference for substrates with a hydroxyl group at the 5' margin of the nick as well as for flapped and recessed DNAs.
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PMID:Efficiency of exonucleolytic action of apurinic/apyrimidinic endonuclease 1 towards matched and mismatched dNMP at the 3' terminus of different oligomeric DNA structures correlates with thermal stability of DNA duplexes. 1648 Dec 27

DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease.
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PMID:Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs. 1668 56

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is multifunctional enzyme. APEI is involved in the DNA base excision repair process (BER). APE1 participates in BER by cleaving the DNA adjacent to the 5' side of an AP site to produce a hydroxyl group at the 3' terminus of an unmodified nucleotide upstream of the nick and a 5' deoxyribose phosphate moiety downstream. In addition to its AP-endonucleolytic function, APE1 possesses 3' phosphodiesterase, 3'-5' exonuclease and 3' phosphatase activities. Independently of being characterized as DNA repair protein, APE1 was identified as redox-factor (Ref-1). Our own and literature data on the role of APE1 additional functions in cell metabolism and on interactions of APE1 with DNA and other proteins that participate in BER are analyzed in this review.
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PMID:[Multifunctional human apurinic/apyrimidinic endonuclease 1: the role of additional functions]. 1768 23

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

A second class II AP endonuclease, APEX2, possesses strong 3'-5' exonuclease and 3'-phosphodiesterase activities but only very weak AP-endonuclease activity. APEX2 associates with proliferating cell nuclear antigen (PCNA), and the progression of S phase of the cell cycle is accompanied by its expression. APEX2-null mice exhibit severe dyslymphopoiesis in thymus as well as moderate dyshematopoiesis and growth retardation. Comparative gene expression profiling of wild-type and APEX2-null mice using an oligonucleotide microarray revealed that APEX2-null thymus has significantly altered gene expression profiles, reflecting its altered populations of thymocytes. Beyond these altered populations, APEX2-null thymus exhibits significant alterations in expression of genes involved in DNA replication, recombination and repair, including Apex1, Exo1 and Fen1 as well as master genes for the DNA damage response, such as E2f1, Chek1, and proapoptotic genes. We therefore examined the extent of DNA strand breakage, and found that both of single-strand breaks detected as comets and double-strand breaks detected as gammaH2AX foci were significantly higher in frequency in most APEX2-null thymocytes compared to wild-type thymocytes. This higher frequency of DNA breaks was accompanied by increased expression of PCNA and increased phosphorylation of p53 at Ser23 and to a lesser extent, at Ser18. The present study clearly demonstrates that APEX2-null lymphocytes have a higher frequency of DNA breaks, indicating that APEX2 may play an important role(s) during their generation and/or repair.
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PMID:Altered gene expression profiles and higher frequency of spontaneous DNA strand breaks in APEX2-null thymus. 1858 82

Human Ape2 protein has 3' phosphodiesterase activity for processing 3'-damaged DNA termini, 3'-5' exonuclease activity that supports removal of mismatched nucleotides from the 3'-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3'-5' exonuclease and 3' phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3'-damaged termini but also by removing 3'-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis.
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PMID:Role of PCNA-dependent stimulation of 3'-phosphodiesterase and 3'-5' exonuclease activities of human Ape2 in repair of oxidative DNA damage. 1944 50

The possibility of interaction of recombinant proteins of human repair DNA polymerase beta with proofreading 3' --> 5'-exonucleases TREX1 and TREX2 was investigated in vitro for the first time. The results of gel filtration analysis show the formation of a complex between 3' --> 5' -exonucleases mTREX1 and hTREX2 and DNA polymerase beta. DNA polymerase activity is shown to increase four-fold in the presence of 3' --> 5'-exonuclease TREX2. The experiments with the use of immunodot and Western blot assays on the binding of DNA-polymerase beta with 3' --> 5'-exonucleases TREX1 and TREX2 immobilized on a nitrocellulose membrane provided additional evidence on the direct association of the above proteins in complexes.
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PMID:[Investigation of the interaction of repair DNA polymerase beta and autonomous 3' --> 5'-exonucleases TREX1 and TREX2]. 2107 63

The base excision repair pathway is largely responsible for the repair of oxidative stress-induced DNA damage. However, it remains unclear how the DNA damage checkpoint is activated by oxidative stress at the molecular level. Here, we provide evidence showing that hydrogen peroxide (H2O2) triggers checkpoint kinase 1 (Chk1) phosphorylation in an ATR [ataxia-telangiectasia mutated (ATM) and Rad3-related]-dependent but ATM-independent manner in Xenopus egg extracts. A base excision repair protein, Apurinic/apyrimidinic (AP) endonuclease 2 (APE2, APN2, or APEX2), is required for the generation of replication protein A (RPA)-bound single-stranded DNA, the recruitment of a checkpoint protein complex [ATR, ATR-interacting protein (ATRIP), and Rad9] to damage sites, and H2O2-induced Chk1 phosphorylation. A conserved proliferating cell nuclear antigen interaction protein box of APE2 is important for the recruitment of APE2 to H2O2-damaged chromatin. APE2 3'-phosphodiesterase and 3'-5' exonuclease activity is essential for single-stranded DNA generation in the 3'-5' direction from single-stranded breaks, referred to as single-stranded break end resection. In addition, APE2 associates with Chk1, and a serine residue (S86) in the Chk1-binding motif of APE2 is essential for Chk1 phosphorylation, indicating a Claspin-like but distinct role for APE2 in ATR-Chk1 signaling. Our data indicate that APE2 plays a vital and previously unexpected role in ATR-Chk1 checkpoint signaling in response to oxidative stress. Thus, our findings shed light on a distinct mechanism of how an ATR-Chk1-dependent DNA damage checkpoint is mediated by APE2 in the oxidative stress response.
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PMID:APE2 is required for ATR-Chk1 checkpoint activation in response to oxidative stress. 2375 35


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