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1.
The conditions for the measurement of linear dichroism (LD) can be adjusted so as to solely reflect the length and the flexibility of DNA. The real-time detection of the EDTA.Fe(2+)-induced oxidative cleavage of double-stranded native and synthetic DNAs was performed using LD. The decrease in the magnitude of the LD at 260 nm, which reflects an increase in the flexibility and a decrease in the length of the DNA, can be described by the sum of two or three exponential curves in relation to the EDTA.Fe(2+) concentration. The fast component was assigned to the cleavage of one of the double strands, inducing an increase in the flexibility, while the other slower component was assigned to the cleavage of the double strand, resulting in the shortening of DNA. The decrease in the magnitude of the LD of poly[d(A-T)(2)] was similar to that of poly[d(I-C)(2)], while that of poly[d(G-C)(2)] was found to be the slowest, indicating that the resistance of poly[d(G-C)(2)] against the Fenton-type reagent was the strongest. This observation suggests that the amine group in the minor groove of the double helix may play an important role in slowing the EDTA.Fe(2+)-induced oxidative cleavage. 相似文献
2.
Fe(2+) can substitute for Mg(2+) in activation of the sarcoplasmic reticulum (SR) ATPase, permitting approximately 25% activity in the presence of Ca(2+). Therefore, we used Fe(2+) to obtain information on the binding sites for Mg(2+) and the Mg(2+)-ATP complex within the enzyme structure. When the ATPase is incubated with Fe(2+) in the presence of H(2)O(2) and/or ascorbate, specific patterns of Fe(2+)-catalyzed oxidation and cleavage are observed in the SR ATPase, depending on its Ca(2+)-bound (E1-Ca(2)) or Ca(2+)-free conformation (E2-TG), as well as on the presence of ATP. The ATPase protein in the E1-Ca(2) state is cleaved efficiently by Fe(2+) with H(2)O(2) and ascorbate assistance, yielding a 70-75 kDa carboxyl end fragment. Cleavage of the ATPase protein in the E2-TG state occurs within the same region, but with a more diffuse pattern, yielding multiple fragments within the 65-85 kDa range. When Fe(2+) catalysis is assisted by ascorbate only (in the absence of H(2)O(2)), cleavage at the same protein site occurs much more slowly, and is facilitated by ATP (or AMP-PNP) and Ca(2+). Amino acid sequencing indicates that protein cleavage occurs at and near Ser346, and is attributed to Fe(2+) bound to a primary Mg(2+) site near Ser346 and neighboring Glu696. In addition, incubation with Fe(2+) and ascorbate produces Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain, as demonstrated by NaB(3)H(4) incorporation and analysis of fragments obtained by extensive trypsin digestion. This oxidation is attributed to bound Fe(2+)-ATP complex, as shown by structural modeling of the Mg(2+)-ATP complex at the substrate site. 相似文献
3.
The reactions of RO(2)* radicals with Fe(H(2)O)(6)(2+) were studied, R[double bond]H; CH(3); CH(2)COOH; CH(2)CN; CH(2)C(CH(3))(2)OH; CH(2)OH; CHCl(2)/CCl(3). All these processes involve the following reactions: Fe(H(2)O)(6)(2+)+RO(2)*<==>(H(2)O)(5)Fe(III)[bond]OOR(2+) K(1) approximately 250 M(-1); (H(2)O)(5)Fe(III)[bond]OOR(2+)+H(3)O(+)/H(2)O-->Fe(H(2)O)(6)(3+)+ROOH+H(2)O/OH(-); (H(2)O)(5)Fe(III)[bond]OOR(2+)+2Fe(H(2)O)(6)(2+)-->3Fe(H(2)O)(6)(3+)+ROH; 2 RO(2)*-->Products; RO(2)*+(H(2)O)(5)Fe(III)[bond]OOR(2+)-->Fe(H(2)O)(6)(2+)+products. The values of k(1) and k(3) [reaction is clearly not an elementary reaction] approach the ligand exchange rate of Fe(H(2)O)(6)(2+), i.e. these reactions follow an inner sphere mechanism and the rate determining step is the ligand exchange step. The rate of reaction is several orders of magnitude faster than that of the Fenton reaction. Surprisingly enough the K(1) values are nearly independent of the redox potential of the radical and are considerably higher than calculated from the relevant redox potentials. These results indicate that the ROO(-) ligands considerably stabilise the Fe(III) complex, this stabilisation is smaller for radicals with electron withdrawing groups which raise the redox potential of the radical but decrease the basicity of the ROO(-) ligands, two effects which seem to nearly cancel each other. Finally, the results clearly indicate that reaction (5) is relatively fast and affects the nature of the final products. The contribution of these reactions to oxidation processes involving 'Fenton-like' processes is discussed. 相似文献
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5.
Marie-Hélène Fouchet Eric Guittet Jean A. H. Cognet Jiří Kozelka Corinne Gauthier Marc Le Bret Karel Zimmermann Jean-Claude Chottard 《Journal of biological inorganic chemistry》1997,2(1):83-92
The structure of the second major adduct formed by the antitumor drug cisplatin with DNA, the intrastand cis–Pt(NH3)2{d(ApG)N7–N7} chelate (A*G*), has been investigated using a double-stranded nonanucleotide, d(CTCA*G*CCTC)-d(GAGGCTGAG), by means of NMR
and molecular modeling. The NMR data allow us to conclude that the oligonucleotide is kinked at the platinated site towards
the major groove in a way similar to that observed elsewhere for the G*G*-crosslink in d(GCCG*G*ATCGC)-d(GCGATCCGGC). The
main difference concerns the position of the thymine T(15) complementary to the platinated adenine A*(4). It remains stacked
on its 5′-neighbor C(14), corresponding to the "model E" described previously, whereas in the G*G*-adduct, the cytosine facing
the 5′-G* was found to oscillate between the 5′-branch ("model E") and the 3′-branch ("model C") of the complementary strand.
Two "E-type" models are presented which account for the particular NOE connectivity and for two remarkable upfield NMR signals:
those of the H2′ proton of the cytidine C(3) 5′ to the A*G* chelate, and of the H3 imino proton of T(15), the base complementary
to A*(4). The former shift is attributed to shielding by the destacked A*(4) base, whereas the latter is accounted for by
a swinging movement of the T(15) base between two positions where the imino Watson-Crick hydrogen bond with A*(4) remains
intact and the amino hydrogen bond is disrupted, or vice versa. Possible implications of the structural difference between
the AG and GG adducts of cisplatin in the mutagenic properties of the two adducts are discussed.
Received: 19 August 1996 / Accepted: 4 November 1996 相似文献
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7.
Mutation in p53 tumor suppressor gene is a hallmark of human cancers. Six major mutational hotspots in p53 contain methylated CpG (mCpG) sites, and C →T transition is the most common mutation at these sites. It was hypothesized that the formation of 5-methylcytosine glycol induced by reactive oxygen species, its spontaneous deamination to thymine glycol and the miscoding property of the latter may account, in part, for the ubiquitous C →T mutation at CpG site. Here, we assessed the kinetics of deamination for two diastereomers of 5-methylcytosine glycol in duplex DNA. Our results revealed that the half-lives for the deamination of the (5S,6S) and (5R,6R) diastereomers of 5-methylcytosine glycol in duplex DNA at 37°C were 37.4 ± 1.6 and 27.4 ± 1.0 h, respectively. The deamination rates were only slightly lower than those for the two diastereomers in mononucleosides. Next, we assessed the formation of 5-methyl-2′-deoxycytidine glycol in the form of its deaminated product, namely, thymidine glycol (Tg), in methyl-CpG-bearing duplex DNA treated with Cu(II)/H2O2/ascorbate. LC-MS/MS quantification results showed that the yield of Tg is similar as that of 5-(hydroxymethyl)-2′-deoxycytidine. Together, our data support that the formation and deamination of 5-methylcytosine glycol may contribute significantly to the C →T transition mutation at mCpG dinucleotide site. 相似文献
8.
Rahman NS Godderz LJ Stray SJ Capra JD Rodgers KK 《The Journal of biological chemistry》2006,281(18):12370-12380
Antibody and T cell receptor genes are assembled from gene segments by V(D)J recombination to produce an almost infinitely diverse repertoire of antigen specificities. Recombination is initiated by cleavage of conserved recombination signal sequences (RSS) by RAG1 and RAG2 during lymphocyte development. Recent evidence demonstrates that recombination can occur at noncanonical RSS sites within Ig genes or at other loci, outside the context of normal lymphocyte receptor gene rearrangement. We have characterized the ability of the RAG proteins to bind and cleave a cryptic RSS (cRSS) located within an Ig V(H) gene segment. The RAG proteins bound with sequence specificity to either the consensus RSS or the cRSS. The RAG proteins nick the cRSS on both the top and bottom strands, thereby bypassing the formation of the DNA hairpin intermediate observed in RAG cleavage of canonical RSS substrates. We propose that the RAG proteins may utilize an alternative mechanism for double-stranded DNA cleavage, depending on the substrate sequence. These results have implications for further diversification of the antigen receptor repertoire as well as the role of the RAG proteins in genomic instability. 相似文献
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Dmitry Namgaladze H Werner Hofer Volker Ullrich 《The Journal of biological chemistry》2002,277(8):5962-5969
The interaction of protein serine/threonine phosphatase calcineurin (CaN) with superoxide and hydrogen peroxide was investigated. Superoxide specifically inhibited phosphatase activity of CaN toward RII (DLDVPIPGRFDRRVSVAAE) phosphopeptide in tissue and cell homogenates as well as the activity of the enzyme purified under reducing conditions. Hydrogen peroxide was an effective inhibitor of CaN at concentrations several orders of magnitude higher than superoxide. Inhibition by superoxide was calcium/calmodulin-dependent. Nitric oxide (NO) antagonized superoxide action on CaN. We provide kinetic and spectroscopic evidence that native, catalytically active CaN has a Fe(2+)-Zn(2+) binuclear center in its active site that is oxidized to Fe(3+)-Zn(2+) by superoxide and hydrogen peroxide. This oxidation is accompanied by a gain of manganese dependence of enzyme activity. CaN isolated by a conventional purification procedure was found in the oxidized, ferric enzyme form, and it became increasingly dependent on divalent cations. These results point to a complex redox regulation of CaN phosphatase activity by superoxide, which is modified by calcium, NO, and superoxide dismutase. 相似文献
12.
Mg(2+) -Responsive riboswitches represent a fascinating example of bifunctional RNAs that sense Mg(2+) ions with high selectivity and autonomously regulate the expression of Mg(2+) -transporter proteins. The mechanism of the mgtA riboswitch is scarcely understood, and a detailed structural analysis is called for to study how this RNA can selectively recognize Mg(2+) and respond by switching between two alternative stem loop structures. In this work, we investigated the structure and Mg(2+) -binding properties of the lower part of the antiterminator loop C from the mgtA riboswitch of Yersinia enterocolitica by solution NMR and report a discrete Mg(2+) -binding site embedded in the AU-rich sequence. At the position of Mg(2+) binding, the helical axis exhibits a distinct kink accompanied by a widening of the major groove, which accommodates the Mg(2+) -binding pocket. An unusually large overlap between two adenine residues on the opposite strands suggests that the bending may be sequence-induced by strong stacking interactions, enabling Mg(2+) to bind at this so-far not described metal-ion binding site. 相似文献
13.
Certain DNA sequences are known to be unusually sensitive to nicking via the Fe2+-mediated Fenton reaction. Most notable are a purine nucleotide followed by three or more G residues, RGGG, and purine nucleotides flanking a TG combination, RTGR. Our laboratory previously demonstrated that nicking in the RGGG sequences occurs preferentially 5' to a G residue with the nicking probability decreasing from the 5' to 3'end of these sequences. Using 1H NMR to characterize Fe2+ binding within the duplex CGAGTTAGGGTAGC/GCTACCCTAACTCG and 7-deazaguanine-containing (Z) variants of it, we show that Fe2+ binds preferentially at the GGG sequence, most strongly towards its 5' end. Substitutions of individual guanines with Z indicate that the high affinity Fe2+ binding at AGGG involves two adjacent guanine N7 moieties. Binding is accompanied by large changes in specific imino, aromatic and methyl proton chemical shifts, indicating that a locally distorted structure forms at the binding site that affects the conformation of the two base pairs 3' to the GGG sequence. The binding of Fe2+ to RGGG contrasts with that previously observed for the RTGR sequence, which binds Fe2+ with negligible structural rearrangements. 相似文献
14.
The origin of the anomalous H8 chemical shifts observed in 1H-NMR spectra of oligonucleotides cross-linked at a GpG sequence with cis-[Pt(NH3)2]2+ has been investigated and clarified. The main contributions that distinguish the H8 resonances of the two platinum-ligating guanines from other GH8 signals and from each other are: (a) the inductive effect of platinum binding which we have recently quantified as a downfield shift of 0.48 +/- 0.07 ppm (M. H. Fouchet, D. Lemaire, J. Kozelka and J.-C. Chottard, unpublished results); (b) the ring-current effect of one GpG guanine on the H8 resonance of the other guanine, which is negative (shielding) for the 5'-H8 and positive (deshielding) for the 3'-H8 in single-stranded adducts, but has the opposite sign in double-stranded adducts; (c) a deshielding polarization effect of the phosphate 5' to the GpG unit. The different signs of the ring-current effects in single-stranded and double-stranded oligonucleotides originate from the orientation of the guanines in the cis-[Pt(NH3)2(Gua)2]2+ moiety (Gua, guanine), which is left-handed helicoidal in single strands and right-handed helicoidal in double strands. In the platinated dinucleotides (cis-[Pt(NH3)2(GpG)]+, cis-[Pt(NH3)2(d(GpG))]+ and cis-[Pt(NH3)2(d(GpG))]), the guanines assume either the left-handed or the right-handed arrangement, depending on the sugar moiety (ribose or deoxyribose), protonation state at N1 and, in the solid state, on crystal forces. This work shows that chemical shifts contain valuable structural information which is complementary to that extracted from correlated spectroscopy and nuclear Overhauser spectroscopy data. 相似文献
15.
Efficient, Mg(2+)-dependent photochemical DNA cleavage by the antitumor quinobenzoxazine (S)-A-62176
The quinobenzoxazines, a group of structural analogues of the antibacterial fluoroquinolones, are topoisomerase II inhibitors that have demonstrated promising anticancer activity in mice. It has been proposed that the quinobenzoxazines form a 2:2 drug-Mg(2+) self-assembly complex on DNA. The quinobenzoxazine (S)-A-62176 is photochemically unstable and undergoes a DNA-accelerated photochemical reaction to afford a highly fluorescent photoproduct. Here we report that the irradiation of both supercoiled DNA and DNA oligonucleotides in the presence of (S)-A-62176 results in photochemical cleavage of the DNA. The (S)-A-62176-mediated DNA photocleavage reaction requires Mg(2+). Photochemical cleavage of supercoiled DNA by (S)-A-62176 is much more efficient that the DNA photocleavage reactions of the fluoroquinolones norfloxacin, ciprofloxacin, and enoxacin. The photocleavage of supercoiled DNA by (S)-A-62176 is unaffected by the presence of SOD, catalase, or other reactive oxygen scavengers, but is inhibited by deoxygenation. The photochemical cleavage of supercoiled DNA is also inhibited by 1 mM KI. Photochemical cleavage of DNA oligonucleotides by (S)-A-62176 occurs most extensively at DNA sites bound by drug, as determined by DNase I footprinting, and especially at certain G and T residues. The nature of the DNA photoproducts, and inhibition studies, indicate that the photocleavage reaction occurs by a free radical mechanism initiated by abstraction of the 4'- and 1'-hydrogens from the DNA minor groove. These results lend further support for the proposed DNA binding model for the quinobenzoxazine 2:2 drug-Mg(2+) complex and serve to define the position of this complex on the minor groove of DNA. 相似文献
16.
Yan-Guo Ren Javier Martínez Anders Virtanen 《The Journal of biological chemistry》2002,277(8):5982-5987
Poly(A)-specific ribonuclease (PARN) is the only mammalian exoribonuclease characterized thus far with high specificity for degrading the mRNA poly(A) tail. PARN belongs to the RNase D family of nucleases, a family characterized by the presence of four conserved acidic amino acid residues. Here, we show by site-directed mutagenesis that these residues of human PARN, i.e. Asp(28), Glu(30), Asp(292), and Asp(382), are essential for catalysis but are not required for stabilization of the PARN x RNA substrate complex. We have used iron(II)-induced hydroxyl radical cleavage to map Fe(2+) binding sites in PARN. Two Fe(2+) binding sites were identified, and three of the conserved acidic amino acid residues were important for Fe(2+) binding at these sites. Furthermore, we show that the apparent dissociation constant ((app)K(d)) values for Fe(2+) binding at both sites were affected in PARN polypeptides in which the conserved acidic amino acid residues were substituted to alanine. This suggests that these residues coordinate divalent metal ions. We conclude that the four conserved acidic amino acids are essential residues of the PARN active site and that the active site of PARN functionally and structurally resembles the active site for 3'-exonuclease domain of Escherichia coli DNA polymerase I. 相似文献
17.
Wei Wang Gil Jun Lee Kyeung Joo Jang Tae Sub Cho Seog K. Kim 《Nucleic acids research》2008,36(14):e85
The conditions for the measurement of linear dichroism (LD) can be adjusted so as to solely reflect the length and the flexibility of DNA. The real-time detection of the EDTA·Fe2+-induced oxidative cleavage of double-stranded native and synthetic DNAs was performed using LD. The decrease in the magnitude of the LD at 260 nm, which reflects an increase in the flexibility and a decrease in the length of the DNA, can be described by the sum of two or three exponential curves in relation to the EDTA·Fe2+ concentration. The fast component was assigned to the cleavage of one of the double strands, inducing an increase in the flexibility, while the other slower component was assigned to the cleavage of the double strand, resulting in the shortening of DNA. The decrease in the magnitude of the LD of poly[d(A-T)2] was similar to that of poly[d(I-C)2], while that of poly[d(G-C)2] was found to be the slowest, indicating that the resistance of poly[d(G-C)2] against the Fenton-type reagent was the strongest. This observation suggests that the amine group in the minor groove of the double helix may play an important role in slowing the EDTA·Fe2+-induced oxidative cleavage. 相似文献
18.
Recently we have demonstrated that hammerhead ribozymes can be fully substituted with 2'-amino pyrimidines without detriment to the catalytic activity, provided that positions 2.2 and/or 2.1 are not modified. We now report on the potential molecular mechanisms by which 2'-amino groups at these positions inhibit the ribozyme cleavage activity. In the presence of Mg(2+), the 2'-amino modification at positions 2.2 and/or 2.1 had no significant effect on substrate binding. Detailed analysis of the ribozyme initial cleavage rates in the presence of various Mg(2+) concentrations indicated that Mg(2+) binding is inhibited by the 2'-amino group at position 2.1. Furthermore, preannealed substrate molecules to the modified ribozyme are not effectively cleaved upon Mg(2+) addition, indicating an alteration of the ribozyme cleavage step. Surprisingly, the cleavage activity of the modified ribozymes was substantially increased when Mg(2+) ions were replaced by the thiophilic Mn(2+) ions, whereas only a moderate cleavage enhancement occurred with its unmodified version. Taken together, our findings indicate that changes in the sugar at position 2.1 alter Mg(2+)-promoting ribozyme cleavage. 相似文献
19.
Chandrashekaran S Saravanan M Radha DR Nagaraja V 《The Journal of biological chemistry》2004,279(48):49736-49740
The characteristic feature of type II restriction endonucleases (REases) is their exquisite sequence specificity and obligate Mg(2+) requirement for catalysis. Efficient cleavage of DNA only in the presence of Ca(2+) ions, comparable with that of Mg(2+), is previously not described. Most intriguingly, KpnI REase exhibits Ca(2+)-dependent specific DNA cleavage. Moreover, the enzyme is highly promiscuous in its cleavage pattern on plasmid DNAs in the presence of Mn(2+) or Mg(2+), with the complete suppression of promiscuous activity in the presence of Ca(2+). KpnI methyltransferase does not exhibit promiscuous activity unlike its cognate REase. The REase binds to oligonucleotides containing canonical and mapped noncanonical sites with comparable affinities. However, the extent of cleavage is varied depending on the metal ion and the sequence. The ability of the enzyme to be promiscuous or specific may reflect an evolutionary design. Based on the results, we suggest that the enzyme KpnI represents an REase evolving to attain higher sequence specificity from an ancient nonspecific nuclease. 相似文献
20.
Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels 总被引:5,自引:0,他引:5
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L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+)-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca(2+) binding of CaM. 相似文献