共查询到20条相似文献,搜索用时 15 毫秒
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We have analyzed the recognition by various repair endonucleases of DNA base modifications induced by three oxidants, viz. [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), a photochemical source of tert-butoxyl radicals, disodium salt of 1,4-etheno-2,3-benzodioxin-1,4-dipropanoic acid (NDPO(2)), a chemical source of singlet oxygen, and riboflavin, a type-I photosensitizer. The base modifications induced by BCBT, which were previously shown to be mostly 7,8-dihydro-8-oxoguanine (8-oxoGua) residues, were recognized by Fpg and Ogg1 proteins, but not by endonuclease IIII, Ntg1 and Ntg2 proteins. In the case of singlet oxygen induced damage, 8-oxoGua accounted for only 35% of the base modifications recognized by Fpg protein. The remaining Fpg-sensitive modifications were not recognized by Ogg1 protein and relatively poor by endonuclease III, but they were relatively good substrates of Ntg1 and Ntg2. In the case of the damage induced by photoexcited riboflavin, the fraction of Fpg-sensitive base modifications identified as 8-oxoGua was only 23%. In contrast to the damage induced by singlet oxygen, the remaining lesions were not only recognized by Ntg1 and Ntg2 proteins and (relatively poor) by endonuclease III, but also by Ogg1 protein. The analysis of the mutations observed after transfection of modified plasmid pSV2gpt into Escherichia coli revealed that all agents induced near exclusively GC-->TA and GC-->CG transversions, the numbers of which were correlated with the numbers of 8-oxoGua residues and Ntg-sensitive modifications, respectively. In conclusion, both singlet oxygen and the type-I photosensitizer riboflavin induce predominantly oxidative guanine modifications other than 8-oxoGua, which most probably give rise to GC-->CG transversions and in which eukaryotic cells are substrates of Ntg1 and Ntg2 proteins. 相似文献
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Cadet J Delatour T Douki T Gasparutto D Pouget JP Ravanat JL Sauvaigo S 《Mutation research》1999,424(1-2):9-21
Modified purine and pyrimidine bases constitute one of the major classes of hydroxyl-radical-mediated DNA damage together with oligonucleotide strand breaks, DNA-protein cross-links and abasic sites. A comprehensive survey of the main available data on both structural and mechanistic aspects of.OH-induced decomposition pathways of both purine and pyrimidine bases of isolated DNA and model compounds is presented. In this respect, detailed information is provided on both thymine and guanine whereas data are not as complete for adenine and cytosine. The second part of the overview is dedicated to the formation of.OH-induced base lesions within cellular DNA and in vivo situations. Before addressing this major point, the main available methods aimed at singling out.OH-mediated base modifications are critically reviewed. Unfortunately, it is clear that the bulk of the chemical and biochemical assays with the exception of the high performance liquid chromatographic-electrochemical detection (HPLC/ECD) method have suffered from major drawbacks. This explains why there are only a few available accurate data concerning both the qualitative and quantitative aspects of the.OH-induced formation of base damage within cellular DNA. Therefore, major efforts should be devoted to the reassessment of the level of oxidative base damage in cellular DNA using appropriate assays including suitable conditions of DNA extraction. 相似文献
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Nikita A. Kuznetsov Christina Bergonzo Arthur J. Campbell Haoquan Li Grigory V. Mechetin Carlos de?los?Santos Arthur P. Grollman Olga S. Fedorova Dmitry O. Zharkov Carlos Simmerling 《Nucleic acids research》2015,43(1):272-281
Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination. 相似文献
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Reactive oxygen species (ROS) damage DNA which appears to represent the major target involved in mutagenesis, carcinogenesis, and aging cell responses. Various DNA modifications are generated by ROS, but 8-hydroxy-2'-deoxyguanosine (8-oxoG) has retained a lot of attention in the last few years. Therefore, numerous methods have been developed to detect and quantify the extent of 8-oxoG in DNA, most of them requiring a significant amount of DNA that might be limiting in the case of biological samples. 8-oxoG is repaired in Escherichia coli by a specific glycosylase, the Fpg (formamidopyrimidine DNA glycosylase) protein, in a reaction that requires a covalent intermediate favored under reducing conditions. We set up a new assay based on the capture of plasmid DNA into sensitized microplate wells. DNA damaged by photoactivation of methylene blue was adsorbed on a polylysine-treated plastic well. Then the Fpg protein was added, allowed to fix on the damage by taking advantage of minimized glycosylase activity at low temperature and the reductive trapping of the covalent intermediate, yielding to a stable DNA-protein interaction. The trapped protein was subsequently recognized by a specific antibody. A secondary antibody coupled with horseradish peroxidase was used to detect the complex and the measurement was carried out by chemiluminescence. This new assay offers various potentialities, specifically in the field of technology of ROS producers. 相似文献
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DNA repair: models for damage and mismatch recognition 总被引:4,自引:0,他引:4
Maintaining the integrity of the genome is critical for the survival of any organism. To achieve this, many families of enzymatic repair systems which recognize and repair DNA damage have evolved. Perhaps most intriguing about the workings of these repair systems is the actual damage recognition process. What are the chemical characteristics which are common to sites of nucleic acid damage that DNA repair proteins may exploit in targeting sites? Importantly, thermodynamic and kinetic principles, as much as structural factors, make damage sites distinct from the native DNA bases, and indeed, in many cases, these are the features which are believed to be exploited by repair enzymes. Current proposals for damage recognition may not fulfill all of the demands required of enzymatic repair systems given the sheer size of many genomes, and the efficiency with which the genome is screened for damage. Here we discuss current models for how DNA damage recognition may occur and the chemical characteristics, shared by damaged DNA sites, of which repair proteins may take advantage. These include recognition based upon the thermodynamic and kinetic instabilities associated with aberrant sites. Additionally, we describe how small changes in base pair structure can alter also the unique electronic properties of the DNA base pair pi-stack. Further, we describe photophysical, electrochemical, and biochemical experiments in which mismatches and other local perturbations in structure are detected using DNA-mediated charge transport. Finally, we speculate as to how this DNA electron transfer chemistry might be exploited by repair enzymes in order to scan the genome for sites of damage. 相似文献
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DNA mismatch repair (MMR) maintains genomic integrity by correction of mispaired bases and insertion-deletion loops. The MMR pathway can also trigger a DNA damage response upon binding of MutSα to specific DNA lesions such as O(6)methylguanine (O(6)meG). Limited information is available regarding cellular regulation of these two different pathways. Within this report, we demonstrate that phosphorylated hMSH6 increases in concentration in the presence of a G:T mismatch, as compared to an O(6)meG:T lesion. TPA, a kinase activator, enhances the phosphorylation of hMSH6 and binding of hMutSα to a G:T mismatch, though not to O(6)meG:T. UCN-01, a kinase inhibitor, decreases both phosphorylation of hMSH6 and binding of hMutSα to G:T and O(6)meG:T. HeLa MR cells, pretreated with UCN-01 and exposed to MNNG, undergo activation of Cdk1 and mitosis despite phosphorylation of Chk1 and inactivating phosphorylation of Cdc25c. These results indicate that UCN-01 may inhibit an alternative cell cycle arrest pathway associated with the MMR pathway that does not involve Cdc25c. In addition, recombinant hMutSα containing hMSH6 mutated at an N-terminal cluster of four phosphoserines exhibits decreased phosphorylation and decreased binding of hMutSα to G:T and O(6)meG:T. Taken together, these results suggest a model in which the amount of phosphorylated hMSH6 bound to DNA is dependent on the presence of either a DNA mismatch or DNA alkylation damage. We hypothesize that both phosphorylation of hMSH6 and total concentration of bound hMutSα are involved in cellular signaling of either DNA mismatch repair or MMR-dependent damage recognition activities. 相似文献
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Cadet J Bourdat AG D'Ham C Duarte V Gasparutto D Romieu A Ravanat JL 《Mutation research》2000,462(2-3):121-128
Base excision repair (BER) is likely to be the main mechanism involved in the enzymatic restoration of oxidative base lesions within the DNA of both prokaryotic and eukaryotic cells. Emphasis was placed in early studies on the determination of the ability of several bacterial DNA N-glycosylases, including Escherichia coli endonuclease III (endo III) and formamidopyrimidine DNA N-glycosylase (Fpg), to recognize and excise several oxidized pyrimidine and purine bases. More recently, the availability of related DNA repair enzymes from yeast and human has provided new insights into the enzymatic removal of several.OH-mediated modified DNA bases. However, it should be noted that most of the earlier studies have involved globally modified DNA as the substrates. This explains, at least partly, why there is a paucity of accurate kinetic data on the excision rate of most of the modified bases. Interestingly, several oxidized pyrimidine and purine nucleosides have been recently inserted into defined sequence oligonucleotides. The use of the latter substrates, together with overexpressed DNA N-glycosylases, allows detailed studies on the efficiency of the enzymatic release of the modified bases. This was facilitated by the development of accurate chromatographic and mass spectrometric methods aimed at measuring oxidized bases and nucleosides. As one of the main conclusions, it appears that the specificity of both endo III and Fpg proteins is much broader than expected a few years ago. 相似文献
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Excision repair of DNA base damage 总被引:4,自引:0,他引:4
P A Cerutti 《Life sciences》1974,15(9):1567-1575
Exposure of cells to exogenous physical and chemical agents can result in damage to the DNA bases. DNA damage can lead to mutation, malignant transformation and cell death and may possibly be involved in cellular aging. Structurally related base modifications are expected to have similar biological effects regardless of the agent responsible for their formation. The biological effects may be a consequence of the local distortion of the DNA conformation by the lesion rather than of the chemical properties of the modified base . It may be useful, therefore, to classify DNA base damage according to their effect on DNA conformation. The elucidation of the structures of the DNA lesions produced in the living cell represents a prerequisite for the correlation of specific lesions with the biological effects and for the study of the cellular repair processes.Excision repair represents an ubiquitous mechanism in cells for the removal of damaged residues from the DNA. The most specific first step in excision repair is the recognition of the damage by an endonuclease followed by incision of the damaged DNA strand in the proximity of the damage. Several “repair endonucleases” have been characterized from bacteria while the search for the corresponding mammalian enzymes is only beginning. The second, probably less specific step, is the exonucleolytic degradation of the damaged portion of the DNA leading to the removal of the damaged residue. In the removal of both cyclobutane-type photodimers and γ-ray products of the 5,6-dihydroxy-dihydrothymine type is accomplished by the 5′→3′ exonuclease associated with polymerase I. All three polymerases appear to participate in the rebuilding of the degraded portion of the DNA. Studies on the corresponding enzymes in mammalian cells have been initiated. The last step of exicison repair involves the sealing of a phosphodiester bond of the DNA backbone and is accomplished by the enzyme polynucleotide ligase in bacterial and mammalian cells. 相似文献
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Valery I. Ivanov 《Molecular biology reports》1976,3(2):181-187
A speculative model based on published sequences to explain the specific binding of RNA polymerase of E. coli to promoters is suggested: {ie181-1}A fragment 24 base pairs long to the left of the initiation site must not contain the G's in the positions indicated by the circles and must contain T's (or G's) in the positions marked by the squares. In most known cases mutual disposition of the circle and square patterns is as shown above. In one case (the fd G3 promoter) the pattern of squares is shifted by 4 base pairs to the right relative to the pattern of universal non-G's (circles). 相似文献
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Junfang Yan Yi Xie Qianjing Zhang Lu Gan Fang Wang Hongyan Li Jing Si Hong Zhang 《Journal of cellular physiology》2019,234(8):13014-13020
Irradiation (IR) can be used to treat cancer by inducing complex and irreparable DNA damage in the cancer cells, which may lead to their apoptotic death. However, little is known about the molecular mechanism of this DNA damage. Here, the non-small-cell lung cancer cell line A549 was treated with either X-ray or carbon ion combined with bleomycin (BLM). The cell survival rate, frequency of double-strand breaks (DSBs), dynamic changes in γH2AX, and p53 binding protein 1 (53BP1), and protein expression of Ku70, Rad51, and XRCC1 were determined by the clone formation assay, agarose gel electrophoresis, immunofluorescence, and western blot analysis. The results showed that the most obvious complex DSBs occurred in the carbon IR + BLM group. The number of γH2AX and 53BP1 foci in the 0.5 hr X-ray IR + BLM group was the highest (p < 0.001) among all the groups. γH2AX foci were detected in the nucleus at 0.5, 1, 2, and 4 hr, but were distributed throughout the cell at 6 hr after IR in the carbon ion IR + BLM group. The expression of Ku70 increased and XRCC1 decreased at 2 and 6 hr after IR. Our data indicate that a DNA damage frequency of 13.4/Mbp is caused by clustered DNA damage and further show a correlation between γH2AX, 53BP1, and XRCC1 levels and the extent of DNA damage. The results of this study provide insights into DNA damage recognition and a rationale for the clinical use of radiotherapy. 相似文献
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Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques. 相似文献
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Nucleotide excision repair (NER) is one of the major DNA repair pathways in eukaryotic cells counteracting genetic changes caused by DNA damage. NER removes a wide set of structurally diverse lesions such as pyrimidine dimers arising upon UV irradiation and bulky chemical adducts arising upon exposure to carcinogens or chemotherapeutic drugs. NER defects lead to severe diseases including some forms of cancer. In view of the broad substrate specificity of NER, it is of interest to understand how a certain set of proteins recognizes various DNA lesions in the context of a large excess of intact DNA. This review focuses on DNA damage recognition and following stages resulting in preincision complex assembly, the key and still most unclear steps of NER. The major models of primary damage recognition and preincision complex assembly are considered. The contribution of affinity labeling techniques in study of this process is discussed. 相似文献
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Khorasanizadeh S 《Current opinion in structural biology》2011,21(6):744-749
Histone tails undergo methylation at their lysines and arginines. These chemical marks act as traffic signals that direct activity of chromatin remodeling complexes to appropriate regions of the genome. A surprisingly diverse group of effector protein modules in chromatin remodeling complexes and their associated factors are involved in the recognition of histone methyllysines. Previous studies generally painted a picture of individual lysines recognized by these protein modules in a 1:1 fashion. However, recent structural studies show more complex interactions where the critical lysines are recognized in pairs, or in the context of nucleosomal DNA, or within the central pore of repeat motifs. These interactions extend our understanding of how histone tail recognition can be enhanced through coupled interactions within a single module or through the cooperation of two different molecules. 相似文献
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Radiation-induced single-strand breaks were found throughout the 172 bp repeat units of African green monkey component alpha DNA. Two kinds of 3'-ends of 5'-32P-labeled restriction fragments were found, as previously described by others. After irradiation in vitro, the yield of single-strand breaks was 4 X 10(-5) breaks/nucleotide/Gy, as determined by analyses in DNA sequencing type gels. Protection from X-ray damage was found when the DNA received 150 Gy in the presence of 2-mercaptoethanol. The results demonstrate a very sensitive quantitative means to study the role of indirect effects of ionizing radiation on strand-break induction and protection at the base sequence level. Component alpha DNA was isolated from irradiated CV-1 cells and was analyzed for single-strand breaks. Under these conditions the frequency of breaks was less than the frequency obtained when purified DNA was irradiated. The methodology is presented because of its relevance to the study of DNA strand breakage in living cells. 相似文献
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Bjørn Dalhus Jon K. Laerdahl Paul H. Backe & Magnar Bjørås 《FEMS microbiology reviews》2009,33(6):1044-1078
Endogenous DNA damage induced by hydrolysis, reactive oxygen species and alkylation modifies DNA bases and the structure of the DNA duplex. Numerous mechanisms have evolved to protect cells from these deleterious effects. Base excision repair is the major pathway for removing base lesions. However, several mechanisms of direct base damage reversal, involving enzymes such as transferases, photolyases and oxidative demethylases, are specialized to remove certain types of photoproducts and alkylated bases. Mismatch excision repair corrects for misincorporation of bases by replicative DNA polymerases. The determination of the 3D structure and visualization of DNA repair proteins and their interactions with damaged DNA have considerably aided our understanding of the molecular basis for DNA base lesion repair and genome stability. Here, we review the structural biochemistry of base lesion recognition and initiation of one-step direct reversal (DR) of damage as well as the multistep pathways of base excision repair (BER), nucleotide incision repair (NIR) and mismatch repair (MMR). 相似文献