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)

Base excision repair (BER) is crucial for development and for the repair of endogenous DNA damage. However, unlike nucleotide excision repair, the regulation of BER is not well understood. Arsenic, a well-established human carcinogen, is known to produce oxidative DNA damage, which is repaired primarily by BER, whilst high doses of arsenic can also inhibit DNA repair. However, the mechanism of repair inhibition by arsenic and the steps inhibited are not well defined. To address this question we have investigated the regulation of DNA polymerase beta (Pol beta) and AP endonuclease (APE1), in response to low, physiologically relevant doses of arsenic. GM847 lung fibroblasts and HaCaT keratinocytes were exposed to sodium arsenite, As(III), and mRNA, protein levels and BER activity were assessed. Both Pol beta and APE1 mRNA exhibited significant dose-dependant down regulation at doses of As(III) above 1 microM. However, at lower doses Pol beta mRNA and protein levels, and consequently, BER activity were significantly increased. In contrast, APE1 protein levels were only marginally increased by low doses of As(III) and there was no correlation between APE1 and overall BER activity. Enzyme supplementation of nuclear extracts confirmed that Pol beta was rate limiting. These changes in BER correlated with overall protection against sunlight UV-induced toxicity at low doses of As(III) and produced synergistic toxicity at high doses. The results provide evidence that changes in BER due to low doses of arsenic could contribute to a non-linear, threshold dose response for arsenic carcinogenesis.
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PMID:Modulation of DNA polymerase beta-dependent base excision repair in cultured human cells after low dose exposure to arsenite. 1825 56

DNA tandem lesions are comprised of two contiguously damaged nucleotides. This subset of clustered lesions is produced by a variety of oxidizing agents, including ionizing radiation. Clustered lesions can inhibit base excision repair (BER). We report the effects of tandem lesions composed of a thymine glycol and a 5'-adjacent 2-deoxyribonolactone (LTg) or tetrahydrofuran abasic site (FTg). Some BER enzymes that act on the respective isolated lesions do not accept the tandem lesion as a substrate. For instance, endonuclease III (Nth) does not excise thymine glycol (Tg) when it is part of either tandem lesion. Similarly, endonuclease IV (Nfo) does not incise L or F when they are in tandem with Tg. Long-patch BER overcomes inhibition by the tandem lesion. DNA polymerase beta (Pol beta) carries out strand displacement synthesis, following APE1 incision of the abasic site. Pol beta activity is enhanced by flap endonuclease (FEN1), which cleaves the resulting flap. The tandem lesion is also incised by the bacterial nucleotide excision repair system UvrABC with almost the same efficiency as an isolated Tg. These data reveal two solutions that DNA repair systems can use to counteract the formation of tandem lesions.
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PMID:DNA tandem lesion repair by strand displacement synthesis and nucleotide excision repair. 1834 Dec 93

Interactions of APE1 (human apurinic/apyrimidinic endonuclease 1) and DNA polymerase beta with various DNA structures imitating intermediates of DNA repair and replication were investigated by gel retardation and photoaffinity labeling. Photoaffinity labeling of APE1 and DNA polymerase beta was accomplished by DNA containing photoreactive group at the 3 -end in mouse embryonic fibroblast (MEF) cell extract or for purified proteins. On the whole, modification efficiency was the same for MEF-extract proteins and for purified APE1 and DNA polymerase beta depending on the nature of the 5 -group of a nick/gap in the DNA substrate. Some of DNA duplexes used in this work can be considered as short-patch (DNA with the 5 -phosphate group in the nick/gap) or long-patch (DNA containing 5 -sugar phosphate or 5 -flap) base excision repair (BER) intermediates. Other DNA duplexes (3 -recessed DNA and DNA with the 5 -hydroxyl group in the nick/gap) have no relation to intermediates forming in the course of BER. As shown by both methods, APE1 binds with the highest efficiency to DNA substrate containing 5 -sugar phosphate group in the nick/gap, whereas DNA polymerase beta binds to DNA duplex with a mononucleotide gap flanked by the 5 -p group. When APE1 and DNA polymerase beta are both present, a ternary complex APE1-DNA polymerase beta-DNA is formed with the highest efficiency with DNA product of APE1 endonuclease activity and with DNA containing 5 -flap or mononucleotide-gapped DNA with 5 -p group. It was found that APE1 stimulates DNA synthesis catalyzed by DNA polymerase beta, and a human X-ray repair cross-complementing group 1 protein (XRCC1) stimulates APE1 3 -5 exonuclease activity on 3 -recessed DNA duplex.
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PMID:Interaction of APE1 and other repair proteins with DNA duplexes imitating intermediates of DNA repair and replication. 1839 60

Escherichia coli endonuclease IV is an archetype for an abasic or apurinic-apyrimidinic endonuclease superfamily crucial for DNA base excision repair. Here biochemical, mutational and crystallographic characterizations reveal a three-metal ion mechanism for damage binding and incision. The 1.10-A resolution DNA-free and the 2.45-A resolution DNA-substrate complex structures capture substrate stabilization by Arg37 and reveal a distorted Zn3-ligand arrangement that reverts, after catalysis, to an ideal geometry suitable to hold rather than release cleaved DNA product. The 1.45-A resolution DNA-product complex structure shows how Tyr72 caps the active site, tunes its dielectric environment and promotes catalysis by Glu261-activated hydroxide, bound to two Zn2+ ions throughout catalysis. These structural, mutagenesis and biochemical results suggest general requirements for abasic site removal in contrast to features specific to the distinct endonuclease IV alpha-beta triose phosphate isomerase (TIM) barrel and APE1 four-layer alpha-beta folds of the apurinic-apyrimidinic endonuclease families.
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PMID:DNA apurinic-apyrimidinic site binding and excision by endonuclease IV. 1840 31

The mammalian AP-endonuclease (APE1) repairs apurinic/apyrimidinic (AP) sites and strand breaks with 3' blocks in the genome that are formed both endogenously and as intermediates during base excision repair. APE1 has an unrelated activity as a redox activator (and named Ref-1) for several trans-acting factors. In order to identify whether any of the seven cysteine residues in human APE1 affects its enzymatic function, we substituted these singly or multiply with serine. The repair activity is not affected in any of the mutants except those with C99S mutation. The Ser99-containing mutant lost affinity for DNA and its activity was inhibited by 10 mM Mg(2+). However, the Ser99 mutant has normal activity in 2 mM Mg(2+). Using crystallographic data and molecular dynamics simulation, we have provided a mechanistic basis for the altered properties of the C99S mutant. We earlier predicted that Mg(2+), with potential binding sites A and B, binds at the B site of wild-type APE1-substrate complex and moves to the A site after cleavage occurs, as observed in the crystal structure. The APE1-substrate complex is stabilized by a H bond between His309 and the AP site. We now show that this bond is broken to destabilize the complex in the absence of the Mg(2+). This effect due to the mutation of Cys99, approximately 16 A from the active site, on the DNA binding and activity is surprising. Mg(2+) at the B site promotes stabilization of the C99S mutant complex. At higher Mg(2+) concentration the A site is also filled, causing the B-site Mg(2+) to shift together with the AP site. At the same time, the H bond between His309 and the AP site shifts toward the 5' site of DNA. These shifts could explain the lower activity of the C99S mutant at higher [Mg(2+)]. The unexpected involvement of Cys99 in APE1's substrate binding and catalysis provides an example of involvement of a residue far from the active site.
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PMID:Unusual role of a cysteine residue in substrate binding and activity of human AP-endonuclease 1. 1843 36

Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5' to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-beta). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-beta, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-beta based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-beta located downstream of APEX1 (3' to the damaged site) and three with pol-beta located upstream of APEX1 (5' to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (approximately -10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available.
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PMID:An AP endonuclease 1-DNA polymerase beta complex: theoretical prediction of interacting surfaces. 1843 3

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifunctional enzyme. In addition to its main AP endonuclease activity, the cleavage of DNA 5' to the AP site, it displays other weak enzymatic activities. One of them is 3'-5' exonuclease activity, which is most effectively pronounced for DNA duplexes containing modified or mismatched nucleotides at the 3' end of the primer chain. There is a presumption that APE1 can correct the DNA synthesis catalyzed by DNA polymerase beta during the base excision repair process. We determined the quantitative parameters of the 3'-5' exonuclease reaction in dependence on the reaction conditions to reveal the detailed mechanism of this process. The kinetic parameters of APE1 exonuclease excision of mismatched dCMP and dTMP from the 3' terminus of single-strand DNA and from photoreactive dCMP analogues applied for photoaffinity modification of proteins and DNA in recombinant systems and cell/nuclear extracts were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.
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PMID:[Quantitative parameters of the 3'-5'-exonuclease reaction of human apurinic/apyrimidinic endonuclease 1 and DNA with single-strand breaks containing dYMP or their modified analogues]. 1852 77

Apurinic/apyrimidinic endonuclease (APE1), a bifunctional AP endonuclease/redox factor, is important in DNA repair and redox signaling, may be associated with radioresistance. Here we investigate whether targeted inhibition of APE1 can sensitize tumor cells to irradiation in vitro and in vivo. We first constructed chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA (Ad5/F35-APE1 siRNA). The infectivity of chimeric Ad5/F35 to LOVO colon cancer cells was greater than that of Ad5. APE1 was strongly expressed and nuclear factor kappaB (NF-kappaB), a downstream molecule of APE1, known as a radioresistance factor, was constitutively active in LOVO cells. Infection of LOVO cells with Ad5/F35-APE1 siRNA resulted in a dose-dependent decrease of APE1 protein and AP endonuclease activity in vitro. Ad5/F35-APE1 siRNA significantly enhanced sensitivity of LOVO cells to irradiation in clonogenic survival assays, associated with increased cell apoptosis. The APE1 expression in LOVO cells was induced by irradiation in a dose-dependent manner, accompanied with the enhancement of DNA-binding activity of NF-kappaB and Ad5/F35-APE1 siRNA effectively inhibited constitutive and irradiation-induced APE1 expression and NF-kappaB activation. In a subcutaneous nude mouse colon cancer model, Ad5/F35-APE1 siRNA (5 x 10(8) IU, intratumoral injection) inhibited the expression of APE1 protein in LOVO xenografts, and significantly enhanced inhibition of tumor growth by irradiation. In conclusion, APE1 may be involved as one of the radioresistance factors, and targeted inhibition of APE1 shows an effective means of enhancing tumor sensitivity to radiotherapy.
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PMID:Chimeric adenoviral vector Ad5/F35-mediated APE1 siRNA enhances sensitivity of human colorectal cancer cells to radiotherapy in vitro and in vivo. 1853 21

APE1/Ref-1 is thought to be a multifunctional protein involved in reduction-oxidation (redox) regulation and base excision DNA repair, and is required for early embryonic development in mice. APE1/Ref-1 has redox activity and AP endonuclease activity, and is able to enhance DNA-binding activity of several transcription factors, including NF-kappaB, AP-1 and p53, through reduction of their critical cysteine residues. However, it remains elusive exactly how APE1/Ref-1 carries out its essential functions in vivo. Here, we show that APE1/Ref-1 not only reduces target transcription factors directly but also facilitates their reduction by other reducing molecules such as glutathione or thioredoxin. The new activity of APE1/Ref-1, termed redox chaperone activity, is exerted at concentration significantly lower than that required for its redox activity and is neither dependent on its redox activity nor on its AP endonuclease activity. We also show evidence that redox chaperone activity of APE1/Ref-1 is critical to NF-kappaB-mediated gene expression in human cells and is mediated through its physical association with target transcription factors. Thus, APE1/Ref-1 may play multiple roles in an antioxidative stress response pathway through its different biochemical activities. These findings also provide new insight into the mechanism of intracellular redox regulation.
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PMID:A new APE1/Ref-1-dependent pathway leading to reduction of NF-kappaB and AP-1, and activation of their DNA-binding activity. 1858 25

The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a byproduct of respiration. ROS-induced DNA lesions, including oxidized bases, abasic (AP) sites, and oxidized AP sites, cause DNA strand breaks and are repaired via the base excision repair (BER) pathway in both the nucleus and mitochondria. Repair of damaged bases and AP sites involving 1-nucleotide incorporation, named single nucleotide (SN)-BER, was observed with mitochondrial and nuclear extracts. During SN-BER, the 5'-phosphodeoxyribose (dRP) moiety, generated by AP-endonuclease (APE1), is removed by the lyase activity of DNA polymerase gamma (pol gamma) and polymerase beta in the mitochondria and nucleus, respectively. However, the repair of oxidized deoxyribose fragments at the 5' terminus after strand break would require 5'-exo/endonuclease activity that is provided by the flap endonuclease (FEN-1) in the nucleus, resulting in multinucleotide repair patch (long patch (LP)-BER). Here we show the presence of a 5'-exo/endonuclease in the mitochondrial extracts of mouse and human cells that is involved in the repair of a lyase-resistant AP site analog via multinucleotide incorporation, upstream and downstream to the lesion site. We conclude that LP-BER also occurs in the mitochondria requiring the 5'-exo/endonuclease and pol gamma with 3'-exonuclease activity. Although a FEN-1 antibody cross-reacting species was detected in the mitochondria, it was absent in the LP-BER-proficient APE1 immunocomplex isolated from the mitochondrial extract that contains APE1, pol gamma, and DNA ligase 3. The LP-BER activity was marginally affected in FEN-1-depleted mitochondrial extracts, further supporting the involvement of an unidentified 5'-exo/endonuclease in mitochondrial LP-BER.
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PMID:Long patch base excision repair in mammalian mitochondrial genomes. 1863 52


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