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Apurinic/apyrimidinic endonucleases initiate the repair of abasic sites produced either spontaneously, from attack of bases by reactive oxygen species or as intermediates during base excision repair. The catalytic properties and crystal structure of Leishmania major apurinic/apyrimidinic endonuclease are described and compared with those of human APE1 and bacterial exonuclease III. The purified enzyme is shown to possess apurinic/apyrimidinic endonuclease activity of the same order as eukaryotic and prokaryotic counterparts and an equally robust 3'-phosphodiesterase activity. Consistent with this, expression of the L. major endonuclease confers resistance to both methyl methane sulphonate and H2O2 in Escherichia coli repair-deficient mutants while expression of the human homologue only reverts methyl methane sulphonate sensitivity. Structural analyses and modelling of the enzyme-DNA complex demonstrates a high degree of conservation to previously characterized homologues, although subtle differences in the active site geometry might account for the high 3'-phosphodiesterase activity. Our results confirm that the L. major's enzyme is a key element in mediating repair of apurinic/apyrimidinic sites and 3'-blocked termini and therefore must play an important role in the survival of kinetoplastid parasites after exposure to the highly oxidative environment within the host macrophage.  相似文献   

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The Ape1 protein initiates the repair of apurinic/apyrimidinic sites during mammalian base excision repair (BER) of DNA. Ape1 catalyzes hydrolysis of the 5'-phosphodiester bond of abasic DNA to create nicks flanked by 3'-hydroxyl and 5'-deoxyribose 5-phosphate (dRP) termini. DNA polymerase (pol) beta catalyzes both DNA synthesis at the 3'-hydroxyl terminus and excision of the 5'-dRP moiety prior to completion of BER by DNA ligase. During BER, Ape1 recruits pol beta to the incised apurinic/apyrimidinic site and stimulates 5'-dRP excision by pol beta. The activities of these two enzymes are thus coordinated during BER. To examine further the coordination of BER, we investigated the ability of Ape1 to modulate the deoxynucleotidyltransferase and 5'-dRP lyase activities of pol beta. We report here that Ape1 stimulates 5'-dRP excision by a mechanism independent of its apurinic/apyrimidinic endonuclease activity. We also demonstrate a second mechanism, independent of Ape1, in which conditions that support DNA synthesis by pol beta also enhance 5'-dRP excision. Ape1 modulates the gap-filling activity of pol beta by specifically inhibiting synthesis on an incised abasic substrate but not on single-nucleotide gapped DNA. In contrast to the wild-type Ape1 protein, a catalytically impaired mutant form of Ape1 did not affect DNA synthesis by pol beta. However, this mutant protein retained the ability to stimulate 5'-dRP excision by pol beta. Simultaneous monitoring of 5'-dRP excision and DNA synthesis by pol beta demonstrated that the 5'-dRP lyase activity lags behind the polymerase activity despite the coordination of these two steps by Ape1 during BER.  相似文献   

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J Pierre  J Laval 《Biochemistry》1980,19(22):5024-5029
Two endonucleases specific for DNA-containing apurinic or apyrimidinic sites (AP-endonucleases A and B) have been isolated from Micrococcus luteus and highly purified. These enzymes have no exonuclease activity. Both AP-endonucleases hydrolyze DNA-containing apurinic or apyrimidinic sites at the 5' end of the lesion, thus generating 3'-hydroxyl and 5'-phosphoryl end groups. DNA-containing pyrimidine dimers, introduced at low doses of UV, are not hydrolyzed, whereas DNA-containing lesions, introduced at high doses of UV or by gamma irradiation are nicked by either AP-endonuclease. During hydrolysis of apurinic DNA, neither of the AP-endonucleases acts as a processive enzyme.  相似文献   

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The human LINE-1 endonuclease (L1-EN) contributes in defining the genomic integration sites of the abundant human L1 and Alu retrotransposons. LINEs have been considered as possible vehicles for gene delivery and understanding the mechanism of L1-EN could help engineering them as genetic tools. We tested the in vitro activity of point mutants in three L1-EN residues--Asp145, Arg155, Ile204--that are key for DNA cleavage, and determined their crystal structures. The L1-EN structure remains overall unaffected by the mutations, which change the enzyme activity but leave DNA cleavage sequence specificity mostly unaffected. To better understand the mechanism of L1-EN, we performed molecular dynamics simulations using as model the structures of wild type EN-L1, of two betaB6-betaB5 loop exchange mutants we have described previously to be important for DNA recognition, of the R155A mutant from this study, and of the homologous TRAS1 endonuclease: all confirm a rigid scaffold. The simulations crucially indicate that the betaB6-betaB5 loop shows an anticorrelated motion with the surface loops betaA6-betaA5 and betaB3-alphaB1. The latter loop harbors N118, a residue that alters DNA cleavage specificity in homologous endonucleases, and implies that the plasticity and correlated motion of these loops has a functional importance in DNA recognition and binding. To further explore how these loops are possibly involved in DNA binding, we docked computationally two DNA substrates to our structure, one involving a flipped-out nucleotide downstream the scissile phosphodiester; and one not. The models for both scenarios are feasible and agree with the hypotheses derived from the dynamic simulations. The reduced cleavage activity we have observed for the I204Y mutant above however, favors the flipped out nucleotide model.  相似文献   

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Repetitive DNA sequences, such as those present in microsatellites and minisatellites, telomeres, and trinucleotide repeats (linked to fragile X syndrome, Huntington disease, etc.), account for nearly 30% of the human genome. These domains exhibit enhanced susceptibility to oxidative attack to yield base modifications, strand breaks, and abasic sites; have a propensity to adopt non-canonical DNA forms modulated by the positions of the lesions; and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge on the repair efficiencies of DNA damage within such repetitive sequences is therefore crucial for understanding the impact of such domains on genomic integrity. In the present study, using strategically designed oligonucleotide substrates, we determined the ability of human apurinic/apyrimidinic endonuclease 1 (APE1) to cleave at apurinic/apyrimidinic (AP) sites in a collection of tandem DNA repeat landscapes involving telomeric and CAG/CTG repeat sequences. Our studies reveal the differential influence of domain sequence, conformation, and AP site location/relative positioning on the efficiency of APE1 binding and strand incision. Intriguingly, our data demonstrate that APE1 endonuclease efficiency correlates with the thermodynamic stability of the DNA substrate. We discuss how these results have both predictive and mechanistic consequences for understanding the success and failure of repair protein activity associated with such oxidatively sensitive, conformationally plastic/dynamic repetitive DNA domains.  相似文献   

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Molecular Biology - Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a dominant role...  相似文献   

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Endonuclease IV is the archetype for a conserved apurinic/apyrimidinic (AP) endonuclease family that primes DNA repair synthesis by cleaving the DNA backbone 5' of AP sites. The crystal structures of Endonuclease IV and its AP-DNA complex at 1.02 and 1.55 A resolution reveal how an alpha8beta8 TIM barrel fold can bind dsDNA. Enzyme loops intercalate side chains at the abasic site, compress the DNA backbone, bend the DNA approximately 90 degrees, and promote double-nucleotide flipping to sequester the extrahelical AP site in an enzyme pocket that excludes undamaged nucleotides. These structures suggest three Zn2+ ions directly participate in phosphodiester bond cleavage and prompt hypotheses that double-nucleotide flipping and sharp bending by AP endonucleases provide exquisite damage specificity while aiding subsequent base excision repair pathway progression.  相似文献   

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Translin is a human single-stranded DNA and RNA binding protein that has been highly conserved in eukaryotic evolution. It consists of eight subunits having a highly helical secondary structure that assemble into a ring. The DNA and the RNA are bound inside the ring. Recently, some of us demonstrated that the human translin specifically binds the single-stranded microsatellite repeats, d(GT)(n), the human telomeric repeats, d(TTAGGG)(n), and the Tetrahymena telomeric repeats, d(GGGGTT)(n). These data suggested that translin might be involved in recombination at d(GT)(n).d(AC)(n) microsatellites and in telomere metabolism. Other data indicated that translin might stimulate binding of telomerase to single-stranded telomeric overhangs by unwinding secondary structures formed by the telomeric repeats. Here we present a circular dichroism (CD) analysis of complexes formed between the human translin and the microsatellite and telomeric oligodeoxynucleotides d(GT)(12) and d(TTAGGG)(5). We report that conformational changes occur in both the translin and the oligodeoxynucleotides upon formation of the complexes. In translin octamers bound to the oligodeoxynucleotide d(GT)(12), the fraction of alpha-helices decreases from approximately 67% to approximately 50%, while the fraction of turns and of the unordered structure increases from approximately 11% to approximately 17% and from approximately 19% to approximately 24%, respectively. In the bound oligodeoxynucleotide d(GT)(12), we observed CD shifts which are consistent with a decrease of base stacking and a putative anti-syn switch of some guanines. The oligodeoxynucleotide d(TTAGGG)(5) formed intramolecular quadruplexes under the conditions of our assays and translin was found to unfold the quadruplexes into structures consisting of a single hairpin and three unwound single-stranded d(TTAGGG) repeats. We suggest that such unfolding could account for the stimulation of telomerase activity by translin mentioned above.  相似文献   

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The major apurinic (AP) DNA-binding protein was purified from a HeLa cell line and from the SV40-transformed cell line AT5BIVA derived from a patient with the repair deficiency syndrome ataxia telangiectasia (AT). This protein appears to be identical with the major cellular apurinic/apyrimidinic endonuclease. The two endonucleases differ in their molecular weight (HeLa, 37,600; AT, 38,900) and their dissociation equilibrium constant for AP sites (HeLa, 7.8 X 10(-11) M; AT, 28.3 X 10(-11) M). These variances might be the consequence of a different post-translational modification. Evidence for this interpretation stems from the observation that the AP DNA binding activity of AP endonuclease, as measured in a glass-fiber filter binding assay, is inactivated upon incubation with snake venom phosphodiesterase and that the AP endonuclease from AT cells in 5-10-fold more sensitive than the HeLa enzyme. For both enzymes, the diesterase treatment leads to the formation of a protein of Mr 35,500 which might be the unmodified precursor of AP endonuclease. The loss of AP DNA binding does not reduce but rather increases the catalytic activity of AP endonuclease when measured at excess substrate concentration.  相似文献   

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An endonuclease activity that acts on alkali-labile lesions in x-irradiated PM2 DNA and recognizes apurinic lesions in heat/acid treated DNA has been partially purified from Drosophila melanogaster embryos and its specific activity monitored throughout early development. The enzyme activity also showed a low level of activity on UV-irradiated DNA. The saturation kinetics observed with both x-irradiated and apurinic PM2 DNA substrates were similar. The endonuclease activity exhibited a broad pH optimum between pH 6 and 8.5 and was almost completely inhibited by 100 mM NaCl, 0.1 mM EDTA, 2 mM CaCl12 and 10 mM NEM. The reaction was not completely dependent on the presence of Mg++cation, but optimum activity was obtained at a concentration of 0.1 mM; concentrations greater than 1 mM Mgs++ were inhibitory. The specific activity of the apurinic endonuclease, partially purified from several stages of embryonic and early larval development, remained the same. Unfertilized eggs exhibited a reduced level of this presumptive repair activity.Abbreviations AP endonucleases Apurinic/apyrimidinic endonucleases  相似文献   

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1-Methyl-9H-pyrido-[3,4-b]indole (harmane) inhibits the apurinic/apyrimidinic (AP) endonuclease activity of the UV endonuclease induced by phage T4, whereas it stimulates the pyrimidine dimer-DNA glycosylase activity of that enzyme. E. coli endonuclease IV, E. coli endonuclease VI (the AP endonuclease activity associated with E. coli exonuclease III), and E. coli uracil-DNA glycosylase were not inhibited by harmane. Human fibroblast AP endonucleases I and II also were only slightly inhibited. Therefore, harmane is neither a general inhibitor of AP endonucleases, nor a general inhibitor of Class I AP endonucleases which incise DNA on the 3'-side of AP sites. However, E. coli endonuclease III and its associated dihydroxythymine-DNA glycosylase activity were both inhibited by harmane. This observation suggests that harmane may inhibit only AP endonucleases which have associated glycosylase activities.  相似文献   

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The aromatic amine 9-amino-ellipticine is a synthetic DNA intercalating compound derived from the antitumor agent ellipticine, which cleaves at very low doses DNA containing apurinic sites by beta-elimination through formation of a Schiff base. This compound has been shown to potentiate the cytotoxic effect of alkylating drugs, such as dimethyl sulfate, in E. coli through a mechanism involving apurinic sites. We have studied the ability of 9-amino-ellipticine to inhibit an enzymatic repair system mimicking base-excision repair, in which E. coli exonuclease III only presents an endonuclease for apurinic/apyrimidinic site activity. 10 microM of 9-amino-ellipticine inhibits 70% of apurinic site repair. Other intercalating agents with similar affinities for DNA do not induce any inhibition. In another system designed for the direct assay of the exonuclease III-induced incisions 5' to AP sites 10 microM of 9-amino-ellipticine inhibits 65% of the endonuclease for apurinic/apyrimidinic site activity of E. coli exonuclease III. The 9-amino-ellipticine-induced formation of a 2',3'-unsaturated deoxyribose and cleavage at the 3' side of the apurinic site, and possible creation of an adduct, as suggested by Bertrand and coworkers (1989), on the 3' position of the deoxyribose seem to strongly inhibit the endonuclease for apurinic/apyrimidinic site activity. 9-Amino-ellipticine appears therefore to be the first small ligand which can inhibit, by an irreversible modification of the substrate, the repair of apurinic sites through the base excision-repair pathway at a pharmacological concentration.  相似文献   

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CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.  相似文献   

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The roles of DNA repair by apurinic/apyrimidinic (AP) endonucleases alone, and together with DNA protection by α/β-type small acid-soluble spore proteins (SASP), in Bacillus subtilis spore resistance to different types of radiation have been studied. Spores lacking both AP endonucleases (Nfo and ExoA) and major SASP were significantly more sensitive to 254-nm UV-C, environmental UV (>280 nm), X-ray exposure, and high-energy charged (HZE)-particle bombardment and had elevated mutation frequencies compared to those of wild-type spores and spores lacking only one or both AP endonucleases or major SASP. These findings further implicate AP endonucleases and α/β-type SASP in repair and protection, respectively, of spore DNA against effects of UV and ionizing radiation.  相似文献   

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Werner syndrome patients are deficient in the Werner protein (WRN), which is a multifunctional nuclear protein possessing 3'-5' exonuclease and ATP-dependent helicase activities. Studies of Werner syndrome cells and biochemical studies of WRN suggest that WRN plays a role in several DNA metabolic pathways. WRN interacts with DNA polymerase beta (pol beta) and stimulates pol beta strand displacement synthesis on a base excision repair (BER) intermediate in a helicase-dependent manner. In this report, we examined the effect of the major human apurinic/apyrimidinic endonuclease (APE1) and of pol beta on WRN helicase activity. The results show that WRN alone is able to unwind several single strand break BER intermediates. However, APE1 inhibits WRN helicase activity on these intermediates. This inhibition is likely due to the binding of APE1 to nicked apurinic/apyrimidinic sites, suggesting that APE1 prevents the promiscuous unwinding of BER intermediates. This inhibitory effect was relieved by the presence of pol beta. A model involving the pol beta-mediated hand-off of WRN protein is proposed based on these results.  相似文献   

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