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)

The cytotoxic effect of the chemotherapeutic drug etoposide (VP-16) is thought to result from its ability to induce DNA damage and thereby to trigger apoptosis. Internucleosomal DNA fragmentation occurs late during apoptosis in many cell types. However, whereas human osteosarcoma cells undergo internucleosomal DNA fragmentation during staurosporine-induced apoptosis, they fail to do so in response to VP-16. Recently, we showed that these cells also do not express the poly(ADP-ribosyl)ation-regulated Ca(2+)- and Mg(2+)-dependent endonuclease DNAS1L3. The possibility that this deficiency underlies the failure of these cells to undergo internucleosomal DNA fragmentation in response to VP-16 was investigated. The proteolytic processing and consequent activation of procaspase-3, cleavage of the inhibitory subunit of DNA fragmentation factor, and the degradation of DNA into 50-kb fragments occurred similarly in osteosarcoma cells exposed to either staurosporine or VP-16. However, the additional processing of the 50-kb DNA fragments to oligonucleosomal fragments was not apparent in the VP-16-treated cells. Ectopic expression of DNAS1L3 conferred on osteosarcoma cells the ability to undergo VP-16-induced internucleosomal DNA fragmentation. Furthermore, expression of DNAS1L3 markedly potentiated the cytotoxic effect of VP-16 in these cells. Both DNAS1L3-mediated and staurosporine-induced internucleosomal DNA fragmentation were Ca(2+) dependent, but only the DNAS1L3-mediated DNA cleavage was blocked by expression of a caspase-3-resistant mutant of poly(ADP-ribose) polymerase-1. The present work results suggest a direct relation between the activity of a chemotherapeutic drug (VP-16) and a specific endonuclease (DNAS1L3). They also indicate that internucleosomal DNA fragmentation plays an active role in apoptosis and that the failure of cancer cells to undergo such DNA degradation may contribute to the development of resistance to chemotherapeutic drugs.
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PMID:The Poly(ADP-ribose) polymerase-1-regulated endonuclease DNAS1L3 is required for etoposide-induced internucleosomal DNA fragmentation and increases etoposide cytotoxicity in transfected osteosarcoma cells. 1215 52

DNA single-strand breaks (SSB) are one of the most frequent DNA lesions produced by reactive oxygen species and during DNA metabolism, but the analysis of cellular responses to SSB remains difficult due to the lack of an experimental method to produce SSB alone in cells. By using human cells expressing a foreign UV damage endonuclease (UVDE) and irradiating the cells with UV through tiny pores in membrane filters, we created SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions. Cellular responses to the SSB were characterized by using antibodies and fluorescence microscopy. Upon UV irradiation, poly(ADP-ribose) synthesis occurred immediately in the irradiated area. Simultaneously, but dependent on poly(ADP-ribosyl)ation, XRCC1 was translocated from throughout the nucleus, including nucleoli, to the SSB. The BRCT1 domain of XRCC1 protein was indispensable for its poly(ADP-ribose)-dependent recruitment to the SSB. Proliferating cell nuclear antigen and the p150 subunit of chromatin assembly factor 1 also accumulated at the SSB in a detergent-resistant form, which was significantly reduced by inhibition of poly(ADP-ribose) synthesis. Our results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB.
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PMID:Spatial and temporal cellular responses to single-strand breaks in human cells. 1274 98

The Escherichia coli MutS and MutL proteins have been conserved throughout evolution, although their combined functions in mismatch repair (MMR) are poorly understood. We have used biochemical and genetic studies to ascertain a physiologically relevant mechanism for MMR. The MutS protein functions as a regional lesion sensor. ADP-bound MutS specifically recognizes a mismatch. Repetitive rounds of mismatch-provoked ADP-->ATP exchange results in the loading of multiple MutS hydrolysis-independent sliding clamps onto the adjoining duplex DNA. MutL can only associate with ATP-bound MutS sliding clamps. Interaction of the MutS-MutL sliding clamp complex with MutH triggers ATP binding by MutL that enhances the endonuclease activity of MutH. Additionally, MutL promotes ATP binding-independent turnover of idle MutS sliding clamps. These results support a model of MMR that relies on two dynamic and redundant ATP-regulated molecular switches.
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PMID:The coordinated functions of the E. coli MutS and MutL proteins in mismatch repair. 1288 8

The roles of ATP binding and hydrolysis by MutS in mismatch repair are poorly understood. MutS E694A, in which Glu-694 of the Walker B motif is substituted with alanine, is defective in hydrolysis of bound ATP and has been reported to support MutL-dependent activation of the MutH d(GATC) endonuclease in a trans DNA activation assay (Junop, M. S., Obmolova, G., Rausch, K., Hsieh, P., and Yang, W. (2001) Mol. Cell 7, 1-12). Because the MutH trans activation assay used in these previous studies was characterized by high background and low efficiency, we have re-evaluated the activities of MutS E694A. In contrast to native MutS, which can be isolated in a nucleotide-free form, purified MutS E694A contains 1.0 mol of bound ATP per dimer equivalent, and substoichiometric levels of bound ADP (0.08-0.58 mol/dimer), consistent with the suggestion that the ADP.MutS.ATP complex comprises a significant fraction of the protein in solution (Bjornson, K. P. and Modrich, P. (2003) J. Biol. Chem. 278, 18557-18562). In the presence of Mg2+, endogenous ATP is hydrolyzed with a rate constant of 0.12 min-1 at 30 degrees C, and hydrolysis yields a protein that displays increased specificity for heteroduplex DNA. As observed with wild type MutS, ATP can promote release of MutS E694A from a mismatch. However, the mutant protein is defective in the methyl-directed, mismatch- and MutL-dependent cis activation of MutH endonuclease on a 6.4-kilobase pair heteroduplex, displaying only 1 to 2% of the activity of wild type MutS. The mutant protein also fails to support normal assembly of the MutS.MutL.DNA ternary complex. Although a putative ternary complex can be observed in the presence of MutS E694A, assembly of this structure displays little if any dependence on a mismatched base pair.
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PMID:Hydrolytically deficient MutS E694A is defective in the MutL-dependent activation of MutH and in the mismatch-dependent assembly of the MutS.MutL.heteroduplex complex. 1450 24

We have evaluated the influence of protein poly(ADP-ribosyl)ation in the relaxation of chromatin by exposing a rat liver polynucleosomal extract to micrococcal nuclease (MNase) digestion. The kinetic susceptibility of polynucleosomes to endonuclease digestion was determined as a function of the time of incubation as well as endonuclease concentration. To validate our assay, we also ran control experiments with protein-free calf thymus DNA as the opposite of polynucleosomal DNA. Rat liver chromatin was also incubated in the absence or presence of exogenously added 200 microM betaNAD(+), the poly(ADP-ribosyl)ation substrate, before MNase digestion. For incubations in the presence of betaNAD(+), the synthesis of polynucleosomal poly(ADP-ribose) was stopped with 1 mM benzamide. After addition of MNase, endonuclease digestion was blocked with EDTA to chelate the Mg(2+) ions needed for enzymatic activation, and the samples were subjected to electrophoresis through 1.5% agarose gels. As expected, a faster degradation of chromatin into oligonucleosomal DNA ladders was observed upon protein poly(ADP-ribosyl)ation when the chromatin extract was preincubated with 200 microM betaNAD(+). Thus, our results are consistent with the conclusion that the covalent poly(ADP-ribosyl)ation of polynucleosomal proteins favors a more "relaxed" or "open" structure, which renders chromatin more susceptible to MNase digestion.
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PMID:Polynucleosomal synthesis of poly(ADP-ribose) causes chromatin unfolding as determined by micrococcal nuclease digestion. 1565 42

Higher order chromatin degradation (HOCD) is a stepwise dismantling of the genome through the excision of chromatin loops and their oligomers at matrix attachment regions (MARs) during the early stages of programmed cell death. Although HOCD ultimately leads to the inactivation of the genome and cell death, a partial HOCD in cells receiving sublethal signals may result in the loss of genetic stability leading to neoplasia, degeneration, and aging. The present study was undertaken to determine the role of protein poly(ADP-ribosyl)ation in HOCD. Nuclei isolated from rat glioma C6 cells were able to carry poly(ADP-ribosyl)ation as assessed by the incorporation of (32)P-NAD(+) into TCA-insoluble fraction. Under the same experimental conditions, millimolar NAD(+) induced rapid HOCD in nuclei. However, while poly(ADP-ribosyl)ation was totally abrogated by specific inhibitor, benzamide, NAD(+)-induced HOCD was unaffected. Benzamide also failed to inhibit HOCD induced by H(2)O(2) exposure in intact cells. These results indicate that HOCD is not mediated through chromatin poly(ADP-ribosyl)ation, and that NAD(+) activates MAR-associated endonuclease or facilitates the access of the enzyme to DNA by other mechanisms. Furthermore, other nucleotides including NADP(+), ATP, UTP, GTP, and CTP were also found to induce HOCD in isolated nuclei indicating that HOCD is controlled by nucleotide-related ligands.
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PMID:Nucleotides induce higher order chromatin degradation. 1631 10

DNA single-strand breaks (SSB) activate poly (ADP-ribose) polymerase 1 (PARP1), which then polymerizes ADP-ribosyl groups on various nuclear proteins, consuming cellular energy. Although PARP1 has a role in repairing SSB, activation of PARP1 also causes necrosis and inflammation due to depletion of cellular energy. Here we show that the major mammalian apurinic/apyrimidinic (AP) endonuclease-1 (APE1), an essential DNA repair protein, binds to SSB and suppresses the activation of PARP1. APE1's high affinity for SSB requires Arg177, which is unique in mammalian APEs. PARP1's binding to the cleaved DNA was inhibited, and PARP1 activation was suppressed by the wild-type APE1, but not by the R177A mutant APE1 protein. Cells transiently transfected with the wild-type APE1 decreased the PARP1 activation after H2O2 treatment, while such suppression did not occur with the expression of the R177A APE1 mutant. These results suggest that APE1 suppresses the activation of PARP1 during the repair process of the DNA damage generated by oxidative stress, which may have an important implication for cells to avoid necrosis due to energy depletion.
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PMID:The human apurinic/apyrimidinic endonuclease-1 suppresses activation of poly(adp-ribose) polymerase-1 induced by DNA single strand breaks. 1673 Aug 71

The histone chaperone SET is required for transcription of chromatin templates by RNA polymerase Pol II (Pol II) in vitro. Here we uncover a positive role for SET in dislodging DEK and PARP1, which restrict access to chromatin in the absence of SET and the PARP1 substrate NAD(+). SET binds chromatin, dissociating DEK and PARP1 to allow transcription in the absence of NAD(+). In the absence of SET, depletion of DEK restores chromatin accessibility to endonuclease but does not permit Mediator recruitment or transcription. In the presence of NAD(+), PARP1 poly(ADP-ribosyl)ates and evicts DEK (and itself) from chromatin to permit Mediator loading and transcription independent of SET. An artificial DEK variant resistant to SET and PARP1 represses transcription, indicating a requirement for DEK removal. Therefore, SET, DEK and PARP1 constitute a network governing access to chromatin by the transcription machinery.
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PMID:SET and PARP1 remove DEK from chromatin to permit access by the transcription machinery. 1752 93

Flap endonuclease-1 (FEN1) is a structure specific endonuclease. The natural substrates of FEN1 are 5'-flap structures formed by three DNA chains one of them has unannealed flapped 5'-end (flap). Flap structures are the intermediates of different processes of DNA metabolism, such as DNA recombination, Okazaki fragment maturation during replication of lagging strand, as well as strand displacement DNA synthesis in base excision repair. FEN1 also possesses 5'-exonuclease activity and newly discovered gap endonuclease activity. FEN1 is known to interact physically and functionally with a number of DNA replication and repair proteins such as the proliferating cell nuclear antigen, helicase/nuclease Dna2, WRN and BLM proteins, replication protein A, apurinic/apyrimidinic endonuclease 1, DNA polymerase beta, poly(ADP-riboso) polymerase 1, high mobility group protein 1, integrase of human immunodeficiency virus, transcription coactivator p300, chromatin proteins, cyclin-dependent kinases (Cdk1, Cdk2, Cyclin A). FEN1 activity is significant for maintaining the integrity of repeat sequences in genome. Recent data suppose the correlation between the abnormality of hFEN1 activity and arising/progression of neurodegenerative and cancer diseases. FEN1 has the dramatic effect on cell growth and development thereby attracting the interest to this enzyme.
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PMID:[Flap endonuclease-1 and its role in the processes of DNA metabolism in eucaryotic cells]. 1870 99

Poly-ADP-ribosylation is a unique post-translational modification that controls various nuclear events such as repair of DNA single-strand breaks. Recently, the protein containing the poly-ADP-ribose (pADPr)-binding zinc-finger (PBZ) domain was shown to be a novel AP endonuclease and involved in a cell cycle checkpoint. Here, we determined the three-dimensional structure of the PBZ domain from Drosophila melanogaster CG1218-PA using NMR spectroscopy. The domain folds into a C2H2-type zinc-finger structure in an S configuration, containing a characteristic loop between the zinc-coordinating cysteine and histidine residues. This is distinct from the structure of other C2H2-type zinc fingers. NMR signal changes that occur when pADPr binds to the PBZ domains from CG1218-PA and human checkpoint with FHA (forkhead-associated) and ring finger (CHFR) and mutagenesis suggest that a surface relatively well conserved among PBZ domains may serve as a major interface with pADPr.
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PMID:Solution structure of a zinc-finger domain that binds to poly-ADP-ribose. 2008 64


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