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1.
The effect of cis-diamminedichloroplatinum (II) (cis-DDP) on the formation of interstrand cross-links in DNA and in DNA and chromatin complex from leukocytes was studied. Following the use of cis-DDP the number of DNA-DNA interstrand cross-links was elevated with the increase of cis-DDP concentration and elongation of reaction time. It was also found that nucleic proteins reduce the quantity of the cis-DDP induced DNA-DNA interstrand cross-links in the DNA in nucleoprotein complex when compared with the links in the isolated DNA. 相似文献
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Localization of low-molecular-weight basic proteins in Bacillus megaterium spores by cross-linking with ultraviolet light. 总被引:6,自引:2,他引:4
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Two low-molecular-weight basic proteins, termed A and B proteins, comprise about 15% of the protein of dormant spores of Bacillus megaterium. Irradiation of intact dormant spores with ultraviolet light results in covalent cross-linking of the A and B proteins to other spore macromolecules. The cross-linked A and B proteins are precipitated by ethanol and can be solubilized by treatment with deoxyribonuclease (75%) or ribonuclease (25%). Irradiation of complexes formed in vitro between deoxyribonucleic acid (DNA) or ribonucleic acid and a mixture of the low-molecular-weight basic proteins from spores also resulted in cross-linking of A and B proteins to nucleic acids. The dose-response curves for formation of covalent cross-links were similar for irradiation of both a protein-DNA complex in vitro and intact spores. However, if irradiation was carried out in vitro under conditions where DNA-protein complexes were disrupted, no covalent cross-links were formed. These data suggest that significant amounts of the low-molecular-weight basic proteins unique to bacterial spores are associated with spore DNA in vivo. 相似文献
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Although the biochemical pathways that repair DNA-protein cross-links have not been clearly elucidated, it has been proposed that the partial proteolysis of cross-linked proteins into smaller oligopeptides constitutes an initial step in removal of these lesions by nucleotide excision repair (NER). To test the validity of this repair model, several site-specific DNA-peptide and DNA-protein cross-links were engineered via linkage at (1) an acrolein-derived gamma-hydroxypropanodeoxyguanosine adduct and (2) an apurinic/apyrimidinic site, and the initiation of repair was examined in vitro using recombinant proteins UvrA and UvrB from Bacillus caldotenax and UvrC from Thermotoga maritima. The polypeptides cross-linked to DNA were Lys-Trp-Lys-Lys, Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, and the 16 kDa protein, T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase. For the substrates examined, DNA incision required the coordinated action of all three proteins and occurred at the eighth phosphodiester bond 5' to the lesion. The incision rates for DNA-peptide cross-links were comparable to or greater than that measured on fluorescein-adducted DNA, an excellent substrate for UvrABC. Incision rates were dependent on both the site of covalent attachment on the DNA and the size of the bound peptide. Importantly, incision of a DNA-protein cross-link occurred at a rate approximately 3.5-8-fold slower than the rates observed for DNA-peptide cross-links. Thus, direct evidence has been obtained indicating that (1) DNA-peptide cross-links can be efficiently incised by the NER proteins and (2) DNA-peptide cross-links are preferable substrates for this system relative to DNA-protein cross-links. These data suggest that proteolytic degradation of DNA-protein cross-links may be an important processing step in facilitating NER. 相似文献
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The review concerns the use of the affinity modification method as an integral part of the modern proteomic analysis to search for and identification of proteins belonging to protein ensembles of DNA repair. Affinity modification is based on the preliminary formation of specific non-covalent complex between the target biopolymer and a reagent (chemically reactive analog of biopolymer or low molecular weight ligand) followed by formation of covalent bond between the reagent and the site of the target, to which the reagent is bound, that ensures the method specificity. This method is most widely and effectively used in the study of structural and functional aspects of protein-nucleic acids interactions. Upon construction of DNA probes, in addition to chemically reactive groups and structural elements involved in specific recognition of DNA by proteins, additional groups that facilitate the subsequent affinity isolation of DNA-protein cross-links, can be introduced into the reagent. The review covers recent examples affinity DNA-reactive probe in combination with mass spectrometric and immunological methods to search for and identification in cell extracts, proteins interacting with apurinic/apyrimidinic sites and the proteins recognizing the cross-links in DNA induced by cisplatin. 相似文献
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Damage to DNA caused by exposure of L929 fibroblasts to ozone was reflected by the generation of strand breaks, DNA inter-strand cross-links and DNA-protein cross-links. Addition of propan-2-ol, a hydroxyl radical scavenger, did not affect the formation of strand breaks. In model experiments it appeared that both purines and pyrimidines were involved in DNA inter-strand and DNA-protein cross-links. 相似文献
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DNA–protein cross-links are formed by various DNA-damaging agents including antitumor platinum drugs. The natures of these ternary DNA–Pt–protein complexes (DPCLs) can be inferred, yet much remains to be learned about their structures and mechanisms of formation. We investigated the origin of these DPCLs and their cellular processing on molecular level using gel electrophoresis shift assay. We show that in cell-free media cisplatin [cis-diamminedichloridoplatinum(II)] forms DPCLs more effectively than ineffective transplatin [trans-diamminedichloridoplatinum(II)]. Mechanisms of transformation of individual types of plain DNA adducts of the platinum complexes into the DPCLs in the presence of several DNA-binding proteins have been also investigated. The DPCLs are formed by the transformation of DNA monofunctional and intrastrand cross-links of cisplatin. In contrast, interstrand cross-links of cisplatin and monofunctional adducts of transplatin are stable in presence of the proteins. The DPCLs formed by cisplatin inhibit DNA polymerization or removal of these ternary lesions from DNA by nucleotide excision repair system more effectively than plain DNA intrastrand or monofunctional adducts. Thus, the bulky DNA–protein cross-links formed by cisplatin represent a more distinct and persisting structural motif recognized by the components of downstream cellular systems processing DNA damage considerably differently than the plain DNA adducts of this metallodrug. 相似文献
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Repair of DNA interstrand cross-links is a complex process critical to which is the identification of sites of damage by specific proteins. We have recently identified the structural protein nonerythroid alpha spectrin (alphaSpIISigma) as a component of a nuclear protein complex in normal human cells which is involved in the repair of DNA interstrand cross-links and have shown that it forms a complex with the Fanconi anemia proteins FANCA, FANCC, and FANCG. Using DNA affinity chromatography, we now show that alphaSpIISigma, present in HeLa cell nuclei, specifically binds to DNA containing psoralen interstrand cross-links and that the FANCA, FANCC, and FANCG proteins are bound to this damaged DNA as well. That spectrin binds directly to the cross-linked DNA has been shown using purified bovine brain spectrin (alphaSpIISigma1/betaSpIISigma1)2. Binding of the Fanconi anemia (FA) proteins to the damaged DNA may be either direct or indirect via their association with alphaSpIISigma. These results demonstrate a role for alpha spectrin in the nucleus as well as a new function for this protein in the cell, an involvement in DNA repair. alphaSpIISigma may bind to cross-linked DNA and act as a scaffold to help in the recruitment of repair proteins to the site of damage and aid in their alignment and interaction with each other, thus enhancing the efficiency of the repair process. 相似文献
9.
P L Gutierrez M S Balachandran Nayar R Nardino P S Callery 《Chemico-biological interactions》1987,64(1-2):23-37
Sodium borohydride reduced diaziquone (AZQ) can cause cross-links between DNA molecules, between DNA and proteins and cause single- and double-strand DNA breaks. In order to understand these effects better, we investigated the reduction of diaziquone by borohydride, and looked at reaction products. We found that a major product was formed during the oxidation of the colorless 2-electron reduced AZQ, and that this product was a monoaziridinyl quinone. We interpret this result to mean that both the leaving aziridine as well as the remaining one can alkylate. This mode of alkylation does not explain cross-links which may occur by a different mechanism requiring simultaneous opening of the aziridine rings. Most of the antitumor activity of borohydride reduced AZQ is probably exerted during the oxidation of the 2-electron reduced AZQ (AZQH2). 相似文献
10.
Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system 总被引:16,自引:0,他引:16
J W Hockensmith W L Kubasek W R Vorachek P H von Hippel 《The Journal of biological chemistry》1986,261(8):3512-3518
Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed. 相似文献
11.
Chiral differentiation of DNA adducts formed by enantiomeric analogues of antitumor cisplatin is sequence-dependent
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1,2-GG intrastrand cross-links formed in DNA by the enantiomeric complexes [PtCl(2)(R,R-2,3-diaminobutane (DAB))] and [PtCl(2)(S,S-DAB)] were studied by biophysical methods. Molecular modeling revealed that structure of the cross-links formed at the TGGT sequence was affected by repulsion between the 5'-directed methyl group of the DAB ligand and the methyl group of the 5'-thymine of the TGGT fragment. Molecular dynamics simulations of the solvated platinated duplexes and our recent structural data indicated that the adduct of [PtCl(2)(R,R-DAB)] alleviated this repulsion by unwinding the TpG step, whereas the adduct of [PtCl(2)(S,S-DAB)] avoided the unfavorable methyl-methyl interaction by decreasing the kink angle. Electrophoretic retardation measurements on DNA duplexes containing 1,2-GG intrastrand cross-links of Pt(R,R-DAB)(2+) or Pt(S,S-DAB)(2+) at a CGGA site showed that in this sequence both enantiomers distorted the double helix to the identical extent similar to that found previously for the same sequence containing the cross-links of the parent antitumor cis-Pt(NH(3))(2)(2+) (cisplatin). In addition, the adducts showed similar affinities toward the high-mobility-group box 1 proteins. Hence, whereas the structural perturbation induced in DNA by 1,2-GG intrastrand cross-links of cisplatin does not depend largely on the bases flanking the cross-links, the perturbation related to GG cross-linking by bulkier platinum diamine derivatives does. 相似文献
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A damage-recognition protein which binds to DNA containing interstrand cross-links is absent or defective in Fanconi anemia, complementation group A, cells. 总被引:2,自引:1,他引:1
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A DNA binding protein with specificity for DNA containing interstrand cross-links induced by 4,5',8-trimethylpsoralen (TMP) plus long wavelength ultraviolet (UVA) light has been identified in normal human chromatin. Protein binding to DNA was determined using a gel mobility shift assay and an oligonucleotide containing a hot spot for formation of psoralen interstrand cross-links. Specificity of the damage-recognition protein for cross-links was demonstrated both by a positive correlation between level of cross-link formation in DNA and extent of protein binding and by effective competition by treated but not undamaged DNA for the binding protein. Chromatin protein extracts from cells from individuals with the genetic disorder, Fanconi anemia, complementation group A (FA-A), which have decreased ability to repair damage produced by TMP plus UVA light, failed to show any protein binding to TMP plus UVA treated DNA. We have previously shown that these chromatin protein extracts contain a DNA endonuclease complex, pI 4.6, which specifically recognizes and incises DNA containing interstrand cross-links and which in FA-A cells is defective in its ability to incise this damaged DNA (Lambert et al. (1992) Mutation Res., 273, 57-71). Together, these findings suggest that the DNA binding protein identified is involved in recognition and repair of DNA interstrand cross-links. 相似文献
13.
A model for radiation-induced cross-linking of DNA and proteins has been developed. It is based on initial formation of free radicals on a DNA base, i.e., thymine, and on an amino acid, i.e., tyrosine. It was demonstrated that interaction of these radicals is highly favored as measured by their kinetics and the cross-linked products. The gas chromatography-mass spectrometry methodology used for the identification of the thymine-tyrosine cross-links is suggested as an experimental approach in the measurements of biological cross-links. 相似文献
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The effect of Shope fibroma virus (SFV) infection on host DNA synthesis was investigated. The cytocidal strain, SFV-I, inhibited the incorporation of [3H]thymidine into nuclear DNA very shortly (2 h) after infection, whereas the noncytocidal strain, SFV-W, did so later (10 h postinfection) and to a lesser extent. Furthermore, a two- to threefold stimulation of host DNA synthesis was recorded in SFV-W-infected cells 3 to 4 h after infection. Since virion-associated nucleases have been implicated in the shutoff of host synthesis, these and other enzymatic activities were measured in purified virion preparations. The SFV strains and vaccinia virus contained equivalent amounts of DNA-dependent RNA polymerase, ATPase, and protein kinase activities. However, in SFV-W the pH 4.5 exonuclease activity was lower than in SFV-I and vaccinia virus, and the level of pH 7.8 endonuclease was almost undetectable. To test whether the lack of endonucleolytic activity had some effect on the removal of the cross-links in the parental DNA that occurs after viral penetration, the fate of the virion SFV DNA was followed. The majority (80%) of the SFV-I and SFV-W DNA molecules extracted after viral adsorption sedimented in alkaline sucrose gradients as cross-linked. After 3 h of infection, 75% of the SFV-I DNA molecules lacked cross-links, whereas 78% of the SFV-W DNA still remained cross-linked. The same results were obtained when the presence of cross-links was tested in restriction fragments. Taken together, these results indicate that virion-associated nucleases are involved in the early shutoff of host DNA synthesis and in the elimination of cross-links from the parental viral DNA. 相似文献
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Rearrangement of interstrand cross-links into intrastrand cross-links in cis-diamminedichloroplatinum(II)-modified DNA.
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In the reaction of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, bifunctional intrastrand and interstrand cross-links are formed. In this work, we show that at 37 degrees C interstrand cross-links (ICL) are labile and rearrange into intrastrand cross-links. The ICL instability was first studied with a 10 base pairs (bp) double-stranded oligonucleotide containing a unique site-specific ICL resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(GC/GC) site by a cis-diammineplatinum(II) residue. The bonds between the platinum and the N7 of guanine residues within the interstrand adduct are cleaved. In 50 mM NaCl or NaClO4, this cleavage results in the formation of monofunctional adducts which subsequently form intrastrand cross-links. One cleavage reaction takes place per cross-linked duplex in either of both DNA strands. Whereas the starting cross-linked 10 bp duplex is hydrogen bonded, the two complementary DNA strands separate after the cleavage of the ICL. Under these conditions, the cleavage reaction is irreversible allowing its rate measurement (t1/2= 29+/-2 h) and closure of monofunctional adducts to intrastrand cross-links occurs within single-stranded DNA. Within a longer cross-linked oligonucleotide (20 bp), ICL are apparently more stable (t1/2= 120+/-12 h) as a consequense of monofunctional adducts closure back to ICL. We propose that the ICL cleavage is reversible in DNA and that these adducts rearrange finally into intrastrand cross-links. Our results could explain an 'ICL unhooking' in previously reported in vivo repair studies [Zhenet al. (1993)Carcinogenesis14, 919-924]. 相似文献
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J P Schouten 《The Journal of biological chemistry》1985,260(17):9916-9928
A method is presented for the detection in crude lysates of subnanogram amounts of proteins covalently bound to a specific nucleic acid sequence. The sensitivity of this method enabled us to study proteins cross-linked to specific DNA and mRNA sequences by irradiation of intact Escherichia coli cells with ultraviolet light. Among the proteins cross-linked to pBR322 DNA, the single strand binding protein, the HU-proteins, and the RNA polymerase beta and sigma subunits were present. Some, but not all proteins were cross-linked to 5-bromodeoxyuridine-substituted DNA more efficiently than to normal DNA. Ribosomal protein S1 is by far the most prominent protein cross-linked to mRNAs. Among the proteins cross-linked in smaller amounts to mRNAs are translation initiation factor IF 1, and at least six proteins of the 30 S ribosomal subunit, among which is S21. No 50 S proteins, nor IF-2, IF-3 or any of the elongation factors could be detected. Some UV-induced nucleic acid-protein cross-links were found to be heat-labile. It is concluded that the method employed may be used to compare the proteins interacting with different mRNAs, as well as single-copy DNA sequences from bacteria and eucaryotes with low complexity genomes. 相似文献
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Genomic and gene-specific DNA interstrand cross-links produced by nitrogen mustard (HN2) were measured in the human tumor cell line Colo320HSR. Following exposures that produced greater than or equal to 1 log cell kill, it was found that HN2-induced DNA interstrand cross-links were produced and processed in a heterogeneous fashion within the genome. Cross-links were detected in the amplified, overexpressed c-myc oncogene, whereas in the weakly expressed N-ras gene and the nontranscribed, high copy number alpha-satellite sequence (of chromosome 20), cross-links were not detected. The cross-links in the c-myc oncogene disappeared more rapidly than total genomic cross-links. These results suggest that HN2-induced DNA interstrand cross-links are produced and processed in the genome in a nonrandom fashion. 相似文献
20.
The efficiency of multiplicity reactivation of T4 particles inactivated by platinum(II) complexes is very low. The same is true for marker rescue and functional survival of genes. This can be at least partly explained by the inability of most inactivated virus particles to introduce their DNA into the host cells as demonstrated by electron microscopy. Conformational changes in the DNA, formation of DNA-DNA and DNA-protein cross-links and the damage of proteins participating in the injection process could be responsible for the phenomenon observed. 相似文献