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1.
Thiolate-hemin complexes as chemical models for cytochrome P-450 have been shown to cause cleavage of DNA. The cleavage of DNA to open-circular and linear forms depended on the structure of thiol ligand and the thiol ligand:hemin ratio at pH 7.8. Complete cleavage of DNA was observed by complexes containing thioglycolate ethylester and mercaptoethanol at 400-600 moles excess of thiol ligand to hemin, those containing cysteine, cysteine methylester and cysteine ethylester at 50-200 moles excess, and those containing mercaptopropionylglycine, glutathione, glutathione dimethylester, penta- and nonapeptides at 5-100 moles excess. Inhibition experiments suggested the involvement of active oxygen species in the cleavage of DNA.  相似文献   

2.
Intramolecular transposition by Tn10   总被引:24,自引:0,他引:24  
H W Benjamin  N Kleckner 《Cell》1989,59(2):373-383
Transposon Tn10 promotes the formation of a circular product containing only transposon sequences. We show that these circles result from an intramolecular transposition reaction in which all of the strand cleavage and ligation events have occurred but newly created transposon/target junctions have not undergone repair. The unligated strand termini at these junctions are those expected according to a simple model in which the target DNA is cleaved by a pair of staggered nicks 9 bp apart, transposon sequences are separated from flanking donor DNA by cleavage at the terminal nucleotides on both strands (at both ends) of the element, and 3' transposon strand ends are ligated to 5' target strand ends. The stability of the unligated junctions suggests that they are protected from cellular processing by transposase and/or host proteins. We propose that the nonreplicative nature of Tn10 transposition is determined by the efficiency with which the nontransferred transposon strand is separated from flanking donor DNA and by the nature of the protein-DNA complexes present at the strand transfer junctions.  相似文献   

3.
I-SceI is a homing endonuclease that specifically cleaves an 18-bp double-stranded DNA. I-SceI exhibits a strong preference for cleaving the bottom strand DNA. The published structure of I-SceI bound to an uncleaved DNA substrate provided a mechanism for bottom strand cleavage but not for top strand cleavage. To more fully elucidate the I-SceI catalytic mechanism, we determined the X-ray structures of I-SceI in complex with DNA substrates that are nicked in either the top or bottom strands. The structures resemble intermediates along the DNA cleavage reaction. In a structure containing a nick in the top strand, the spatial arrangement of metal ions is similar to that observed in the structure that contains uncleaved DNA, suggesting that cleavage of the bottom strand occurs by a common mechanism regardless of whether this strand is cleaved first or second. In the structure containing a nick in the bottom strand, a new metal binding site is present in the active site that cleaves the top strand. This new metal and a candidate nucleophilic water molecule are correctly positioned to cleave the top strand following bottom strand cleavage, providing a plausible mechanism for top strand cleavage.  相似文献   

4.
A bis-peptide nucleic acid (PNA)-anthraquinone imide (AQI) conjugate has been synthesized and shown to form strand invasion complexes with a duplex DNA target. The two arms of the bis-PNA each consist of five consecutive thymine residues and are linked by a flexible, hydrophilic spacer. Probing with potassium permanganate reveals that the bis-PNA complexes to duplex DNA at A5.T5sites with local displacement of the T5DNA strand. The 5 bp sequence targeted by the PNA is the shortest strand invasion complex reported to date. Irradiation of the strand invasion complex results in asymmetric cleavage of the displaced strand, with more efficient cleavage at the 3'-end of the loop. This result indicates that the bis-PNA binds to the DNA such that the C-terminal T5sequence forms the strand invasion complex, leaving the N-terminal T5sequence to bind by triplex formation, thereby placing the AQI closer to the 3'-end of the displaced strand, consistent with the observed photocleavage pattern. The ability of the PNA to directly report its binding site by photoinduced cleavage could have significant utility in mapping the secondary and tertiary structure of nucleic acids.  相似文献   

5.
Gu F  Xi Z  Goldberg IH 《Biochemistry》2000,39(16):4881-4891
Bulge structures in nucleic acids are of general biological significance and are potential targets for therapeutic drugs. It has been shown in a previous study that thiol-activated neocarzinostatin chromophore is able to cleave duplex DNA selectively at a position opposite a single unpaired cytosine or thymine base on the 3' side. In this work, we studied in greater detail the nature of this type of cleavage and the basis for the selectivity of the bulge site cleavage over the usual strand cleavage at a T site in the duplex region by using duplexes containing an internal control and a bulge, which is composed of different types and number of bases. Experimental results indicated that the bulge-induced cleavage is initiated by 5' hydrogen abstraction and is greatly affected by the base composition of the bulge. A single-base bulge, especially when containing a purine, yields higher efficiency and greater selectivity for the bulge-induced cleavage. In particular, a single adenine base gives rise to the highest cleavage yield and provides over 20 times greater selectivity for cleavage at the bulge site compared with the internal control site in duplexes. The binding dissociation constants of postactivated drug for a stem-loop structure containing a one- or two-base bulge in the stem, measured by fluorescence quenching, show that the binding is about 3-4 times stronger for bulge-containing duplexes than for perfect hairpin duplexes. For RNA.DNA hybrid duplexes, where the DNA is the target strand and the RNA is the bulge-containing strand, bulge-induced cleavage was observed, although at low yield. On the other hand, when RNA is the nonbulge strand, no bulge-induced cleavage was found. When the reaction is performed in the absence of oxygen, the major product is a covalent adduct, and it is at the same location as the cleavage site under aerobic conditions.  相似文献   

6.
Aberration of eukaryotic topoisomerase I catalysis leads to potentially recombinogenic pathways by allowing the joining of heterologous DNA strands. Recently, a new ligation pathway (flap ligation) was presented for vaccinia virus topoisomerase I, in which blunt end cleavage complexes ligate the recessed end of duplex acceptors having a single-stranded 3'-tail. This reaction was suggested to play an important role in the repair of topoisomerase I-induced DNA double-strand breaks. Here, we characterize flap ligation mediated by human topoisomerase I. We demonstrate that cleavage complexes containing the enzyme at a blunt end allow invasion of a 3'-acceptor tail matching the scissile strand of the donor, which facilitates ligation of the recessed 5'-hydroxyl end. However, the reaction was strictly dependent on the length of double-stranded DNA of the donor complexes, and longer stretches of base-pairing inhibited strand invasion. The stabilization of the DNA helix was most probably provided by the covalently bound enzyme itself, since deleting the N-terminal domain of human topoisomerase I stimulated flap ligation. We suggest that stabilization of the DNA duplex upon enzyme binding may play an important role during normal topoisomerase I catalysis by preventing undesired strand transfer reactions. For flap ligation to function in a repair pathway, factors other than topoisomerase I, such as helicases, would be necessary to unwind the DNA duplex and allow strand invasion.  相似文献   

7.
Copper–phenanthroline complexes oxidatively damage and cleave nucleic acids. Copper bis-phenanthroline and copper complexes of mono- and bis-phenanthroline conjugates are used as research tools for studying nucleic acid structure and binding interactions. The mechanism of DNA oxidation and cleavage by these complexes was examined using two copper–phenanthroline conjugates of the sequence-specific binding molecule, distamycin. The complexes contained either one or two phenanthroline units that were bonded to the DNA-binding domain through a linker via the 3-position of the copper ligand. A duplex containing independently generated 2-deoxyribonolactone facilitated kinetic analysis of DNA cleavage. Oxidation rate constants were highly dependent upon the ligand environment but rate constants describing elimination of the alkali-labile 2-deoxyribonolactone intermediate were not. Rate constants describing DNA cleavage induced by each molecule were 11–54 times larger than the respective oxidation rate constants. The experiments indicate that DNA cleavage resulting from β-elimination of 2-deoxyribonolactone by copper–phenanthroline complexes is a general mechanism utilized by this family of molecules. In addition, the experiments confirm that DNA damage mediated by mono- and bis-phenanthroline copper complexes proceeds through distinct species, albeit with similar outcomes.  相似文献   

8.
A unique reaction for type II DNA topoisomerase is its cleavage of a pair of DNA strands in concert. We show however, that in a reaction mixture containing a molar excess of EDTA over Mg2+, or when Mg2+ is substituted by Ca2+, Mn2+, or Co2+, the enzyme cleaves only one rather than both strands. These results suggest that the divalent cations may play an important role in coordinating the two subunits of DNA topoisomerase II during the strand cleavage reaction. The single strand and the double strand cleavage reactions are similar in the following aspects: both require the addition of a protein denaturant, can be reversed by low temperature or high salt, and a topoisomerase II molecule is attached covalently to the 5' phosphoryl end of each broken DNA strand. Furthermore, the single strand cleavage sites share a similar sequence preference with double strand cleavage sites. There is, however, a strand bias for the single strand cleavage reaction. We show also that under single strand cleavage conditions, topoisomerase II still possesses a low level of double strand passage activity: it can introduce topological knots into both covalently closed or nicked DNA rings, and change the linking number of a plasmid DNA by steps of two. The implication of this observation on the sequential cleavage of the two strands of the DNA duplex during the normal DNA double strand passage process catalyzed by type II DNA topoisomerases is discussed.  相似文献   

9.
Studies in animal models have indicated that dietary isothiocyanates (ITCs) exhibit cancer preventive activities through carcinogen detoxification-dependent and -independent mechanisms. The carcinogen detoxification-independent mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells (e.g. through suppression of IκB kinase and nuclear factor κB as well as other proposed mechanisms). In the current studies we show that ITC-induced apoptosis of oncogene-transformed cells involves thiol modification of DNA topoisomerase II (Top2) based on the following observations. 1) siRNA-mediated knockdown of Top2α in both SV40-transformed MEFs and Ras-transformed human mammary epithelial MCF-10A cells resulted in reduced ITC sensitivity. 2) ITCs, like some anticancer drugs and cancer-preventive dietary components, were shown to induce reversible Top2α cleavage complexes in vitro. 3) ITC-induced Top2α cleavage complexes were abolished by co-incubation with excess glutathione. In addition, proteomic analysis revealed that several cysteine residues on human Top2α were covalently modified by benzyl-ITC, suggesting that ITC-induced Top2α cleavage complexes may involve cysteine modification. Interestingly, consistent with the thiol modification mechanism for Top2α cleavage complex induction, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, was shown to induce Top2α cleavage complexes. In the aggregate, our results suggest that thiol modification of Top2α may contribute to apoptosis induction in transformed cells by ITCs.  相似文献   

10.
11.
Topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus (Sso topo III) is optimally active in DNA relaxation at 75 degrees C. We report here that Sso topo III-catalysed DNA cleavage and religation differed significantly in temperature dependence: the enzyme was most active in cleaving ssDNA containing a cleavage site at 25-50 degrees C, but was efficient in rejoining the cleaved DNA strand only at higher temperatures (e.g. > or = 45 degrees C). The failure of Sso topo III to rejoin the cleaved DNA strand efficiently appeared to be responsible for the inability of the enzyme to relax negatively supercoiled DNA at low temperature (e.g. 25 degrees C). Intriguingly, Sso topo III facilitated DNA annealing although it showed higher affinity for ssDNA than for dsDNA. Religation of the DNA strand cleaved by Sso topo III was drastically enhanced when the DNA was allowed to anneal to a complementary non-cleaved oligonucleotide, presumably as a result of destabilization of the interaction between the enzyme and the cleaved strand through the formation of duplex DNA. A region in the non-cleaved strand corresponding to a sequence containing six bases on the 5' side and two bases on the 3' side of the cleavage site in the cleaved strand was crucial to the annealing-promoted religation. However, the annealing-promoted religation was relatively insensitive to mismatches in this region and the region conserved for oligonucleotide cleavage, except for that at the 5' end of the broken strand. These results suggest that Sso topo III is well suited for a role in DNA rewinding, whether it leads to homoduplex or heteroduplex formation.  相似文献   

12.
The anticancer drug, bleomycin, causes both single and double strand scission of duplex DNA in vitro, with double strand scission occurring in excess of that expected from the random accumulation of single strand nicks. The mechanism of the preferential double strand scission of DNA by bleomycin has been investigated through the synthesis of a series of double hairpin and linear oligonucleotides designed to contain a single nick-like structure at a defined site to serve as models of bleomycin-damaged duplex DNA. The 3' and/or 5' hydroxyls flanking the nick have been phosphorylated to model the increased negative charge at a bleomycin-generated nick. The ability of bleomycin to cleave the intact strand opposite the nick was then determined by autoradiography. The results demonstrate that phosphorylation at either the 3' or 5' hydroxyl, and especially when both sites are phosphorylated, strongly enhances selective cleavage by bleomycin of the opposite strand. These experiments indicate that bleomycin-mediated double strand scission is a form of self-potentiation in which the high affinity of bleomycin for the initially generated nicked sites leads to a greatly enhanced probability of scission of the strand opposite those sites.  相似文献   

13.
J S Vyle  B A Connolly  D Kemp  R Cosstick 《Biochemistry》1992,31(11):3012-3018
Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-diisopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.  相似文献   

14.
To investigate interactions between proteins participating in the long-patch pathway of base excision repair (BER), DNA duplexes with flap strand containing modifications in sugar phosphate backbone within the flap-forming oligonucleotides were designed. When the flap-forming oligonucleotide consisted of two sequences bridged by a decanediol linker located in the flap strand near the branch point, the efficiency and position of cleavage by flap endonuclease 1 (FEN1) differed from those for natural flap. The cleavage rate of chimeric structure by FEN1 was lower than that of a normal substrate. When we introduced the second modification in the flap-forming oligonucleotide, the cleavage rate decreased significantly. To estimate efficiency of recognition and processing of the chimeric structures by BER proteins, we studied the rate of DNA synthesis by DNA polymerase beta (Pol beta) and the rate of nucleotide excision at the 3'-end of the initiating primer by apurinic/apyrimidinic endonuclease 1 (APE1) compared with those for the natural DNA duplexes. Efficiency of strand-displacement DNA synthesis catalyzed by Pol beta was shown to be higher for flap structures containing non-nucleotide linkers. The chimeric structures were processed by the 3'-exonuclease activity of APE1 with efficiency lower than that for a normal flap structure. Thus, DNA duplexes with modifications in sugar phosphate backbone can be used to mimic intermediates of the long-patch pathway of BER in reconstituted systems containing FEN1. Based on chimeric and natural oligonucleotides, photoreactive DNA structures were designed. The photoreactive dCMP moiety was introduced into the 3'-end of DNA primer via the activity of Pol beta. The photoreactive DNA duplexes--3'-recessed DNA, nicked DNA, and flap structures containing natural and chimeric oligonucleotides--were used for photoaffinity labeling of BER proteins.  相似文献   

15.
M J Robinson  N Osheroff 《Biochemistry》1991,30(7):1807-1813
The post-strand-passage DNA cleavage/religation equilibrium of Drosophila melanogaster topoisomerase II was examined. This was accomplished by including adenyl-5'-yl imidodiphosphate, a nonhydrolyzable ATP analogue which supports strand passage but not enzyme turnover, in assays. Levels of post-strand-passage enzyme-mediated DNA breakage were 3-5 times higher than those generated by topoisomerase II prior to the strand-passage event. This finding correlated with a decrease in the apparent first-order rate of topoisomerase II mediated DNA religation in the post-strand-passage cleavage complex. Since previous studies demonstrated that antineoplastic drugs stabilize the pre-strand-passage cleavage complex of topoisomerase II by impairing the enzyme's ability to religate cleaved DNA [Osheroff, N. (1989) Biochemistry 28, 6157-6160; Robinson, M.J., & Osheroff, N. (1990) Biochemistry 29, 2511-2515], the effects of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide on the enzyme's post-strand-passage DNA cleavage complex were characterized. Both drugs stimulated the ability of topoisomerase II to break double-stranded DNA after strand passage. As determined by two independent assay systems, m-AMSA and etoposide stabilized the enzyme's post-strand-passage DNA cleavage complex primarily by inhibiting DNA religation. These results strongly suggest that both the pre- and post-strand-passage DNA cleavage complexes of topoisomerase II serve as physiological targets for these structurally disparate antineoplastic drugs.  相似文献   

16.
The neocarzinostatin chromophore causes double-strand damage at AGC sequences on DNA by concomitant 1'-oxidation at C and 5'-oxidation at the T on the complementary strand. The extent of this damage is dependent upon the structure of the thiol used for activation. Deuterium isotope effects suggest that this dependence on thiol structure may be due to internal quenching of one radical site of the activated chromophore by the hydrogen atoms of the thiol sidechain. The 12-mer d[GCAAGCGCTTGC] is treated with the neocarzinostatin chromophore and either sodium thioglycolate or [2-2H2]-thioglycolate, and the distribution of strand breaks is determined by gel electrophoresis. Two isotope effects are noted: an overall sequence-independent effect in which deuterated thioglycolate increases total strand damage by a factor of 2, and a sequence-specific effect by which deuteration increases the proportion of alkali-sensitive strand damage at C6 by an additional factor of 1.5. Methyl thioglycolate shows essentially identical behavior to that of thioglycolate anion, ruling out electrostatic effects as major contributors to the effect of thiol structure on the mode of DNA damage observed. A model for NCSC action consistent with these results is discussed.  相似文献   

17.
The present study deals with the binding and cleavage by EcoRII endonuclease of concatemer DNA duplexes containing EcoRII recognition sites (formula; see text) in which dT is replaced by dU or 5-bromodeoxyuridine, or 5'-terminal dC in the dT-containing strand is methylated at position 5. The enzyme molecule is found to interact with the methyl group of the dT residue of the DNA recognition site and to be at least in proximity to the H5 atom of the 5'-terminal dC residue in dT-containing strand of this site. Modification of any of these positions exerts an equal effects on the cleavage of both DNA strands. Endonuclease EcoRII was found to bind the substrate specifically. At the same time modification of the bases in recognized sequence may result in the formation of unproductive, though stable, enzyme-substrate complexes.  相似文献   

18.
Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mm Mg(2+) in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg(2+)) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg(2+) results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg(2+) and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg(2+) levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.  相似文献   

19.
The RF IV form of M13 DNA was synthesized enzymatically in vitro, using the viral (+)strand as template, to contain phosphorothioate-modified internucleotidic linkages of the Rp configuration on the 5' side of every base of a particular type in the newly-synthesized (-)strand. Twenty nine restriction enzymes were then tested for their reactions with the appropriate modified DNA types having a phosphorothioate linkage placed exactly at the cleavage site(s) of these enzymes in the (-)strand. Eleven of the seventeen restriction enzymes tested that had recognition sequences of five bases or more could be used to convert the phosphorothioate DNA entirely into the nicked form, either by simply allowing the reaction to go to completion with excess enzyme (Ava I, Ava II, Ban II, Hind II, Nci I, Pst I or Pvu I) or by stopping the reaction at the appropriate time before the nicked DNA is linearized (Bam HI, Bgl I, Eco RI or Hind III). Only modification of the exact cleavage site in the (-)strand could block linearization by the first class of enzymes. The results presented imply that the restriction enzyme-directed nicking of phosphorothioate M13 DNA occurs exclusively in the (+)strand.  相似文献   

20.
We have probed the contacts between EcoRI endonuclease and the central phosphate of its recognition site GAApTTC, using synthetic oligonucleotides containing single stereospecific Rp- or Sp-phosphorothioates (Ps). These substitutions produce subtle stereospecific effects on EcoRI endonuclease binding and cleavage. An Sp-Ps substitution in one strand of the DNA duplex improves binding free energy by -1.5 kcal/mol, whereas the Rp-Ps substitution has an unfavorable effect (+0.3 kcal/mol) on binding free energy. These effects derive principally from changes in the first order rate constants for dissociation of the enzyme-DNA complexes. The first order rate constants for strand scission are also affected, in that a strand containing Sp-Ps substitution is cleaved 2 to 3 times more rapidly than a strand containing a normal prochiral phosphate, whereas a strand containing Rp-Ps substitution is cleaved about 3 times slower than normal. As a result, single-strand substitutions produce pronounced asymmetry in the rates of cleavage of the two DNA strands, and this effect is exaggerated in an Rp,Sp-heteroduplex. Ethylation-interference footprinting indicates that none of the Ps substitutions cause any major change in contacts between endonuclease and DNA phosphates. When an Sp-Ps localizes P = O in the DNA major groove, a hydrogen-bonding interaction with the backbone amide-NH of Gly116 of the endonuclease is improved relative to that with a prochiral phosphate having intermediate P-O bond order and delocalized charge.  相似文献   

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