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Jean Cadet

Publications and source records attributed to Jean Cadet.

At least 19 recordsLinked to original sources
PubMed2006

Radiation-induced formation of DNA intrastrand crosslinks between thymine and adenine bases: A theoretical approach.

The role of local geometric and stereo-electronic effects in tuning the radiation-induced formation of intrastrand crosslinks between adenine and thymine has been analyzed by a computational approach rooted in density functional theory. Our study points out that together with steric accessibility, stereo-electronic effects play a major role in determining the reaction mechanism and the observed predominance of the thymine-adenine lesion over the opposite sequence isomer.

Adenine↗
PubMed2006

Guanine-thymine intrastrand cross-linked lesion containing oligonucleotides: from chemical synthesis to in vitro enzymatic replication.

An intrastrand cross-link lesion, in which two neighboring nucleobases are covalently tethered, has been site-specifically synthesized into defined sequence oligonucleotides in order to perform in vitro replication studies using either bacterial replicative or translesional synthesis polymerases. The investigated tandem base lesion that involves a cross-link between the methylene group of thymine and the C8 of an adjacent guanine residue has been prepared by UV-photolysis under anaerobic condition of the photolabile precursor 5-(phenylthiomethyl)-2'-deoxyuridine that has been site-specifically incorporated into a 9-mer oligonucleotide. After ligation, the lesion-containing modified oligonucleotide was used as a DNA template in primer extension reactions catalyzed by several DNA polymerases including the fragment Klenow exo-(Kf-) of E. coli polymerase I, the Thermus aquaticus polymerase (Taq pol) and the E. coli translesional DNA polymerase Pol IV (dinB). It was found that the primer extension reaction was stopped after the incorporation of the correct nucleotide dAMP opposite the 3'-thymine residue of guanine(C8-CH2) thymine lesion by Kf- and Pol IV; however it was noted that the efficiency of the nucleotide incorporation was reduced. In contrast, the Taq polymerase was totally blocked at the nucleotide preceding the tandem lesion. These results are strongly suggestive that the present intrastrand cross-link lesion, if not repaired, would constitute a blocking lesion for prokaryotic DNA polymerases, being likely lethal for the cell.

Animals↗
PubMed2006

Cyclobutane pyrimidine dimers are predominant DNA lesions in whole human skin exposed to UVA radiation.

Solar UV radiation is the most important environmental factor involved in the pathogenesis of skin cancers. The well known genotoxic properties of UVB radiation (290-320 nm) mostly involve bipyrimidine DNA photoproducts. In contrast, the contribution of more-abundant UVA radiation (320-400 nm) that are not directly absorbed by DNA remains poorly understood in skin. Using a highly accurate and quantitative assay based on HPLC coupled with tandem mass spectrometry, we determined the type and the yield of formation of DNA damage in whole human skin exposed to UVB or UVA. Cyclobutane pyrimidine dimers, a typical UVB-induced DNA damage, were found to be produced in significant yield also in whole human skin exposed to UVA through a mechanism different from that triggered by UVB. Moreover, the latter class of photoproducts is produced in a larger amount than 8-oxo-7,8-dihydro-2'-deoxyguanosine, the most common oxidatively generated lesion, in human skin. Strikingly, the rate of removal of UVA-generated cyclobutane pyrimidine dimers was lower than those produced by UVB irradiation of skin. Finally, we compared the formation yields of DNA damage in whole skin with those determined in primary cultures of keratinocytes isolated from the same donors. We thus showed that human skin efficiently protects against UVB-induced DNA lesions, whereas very weak protection is afforded against UVA. These observations emphasize the likely role played by the UVA-induced DNA damage in skin carcinogenesis and should have consequences for photoprotection strategies.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2006

Formation of isodialuric acid lesion within DNA oligomers via one-electron oxidation of 5-hydroxyuracil: characterization, stability and excision repair.

5-Hydroxyuracil is a major oxidized nucleobase that can be generated by the action of (*)OH radical and one-electron oxidants. The latter modified base that exhibits a low ionization potential is highly susceptible to further degradation upon exposure to various oxidants. Emphasis was placed in this work on the formation and characterization of one-electron oxidation products of 5-hydroxyuracil within DNA fragments of defined sequence. For this purpose, 5-hydroxyuracil containing single- and double-stranded oligonucleotides of various lengths were synthesized and then exposed to the oxidizing action of iridium salts. Isodialuric acid was found to be formed almost quantitatively by a one-electron oxidation mechanism for which relevant information was inferred from a freeze-quenched ESR study. Information on the stability of isodialuric acid thus formed and its conversion products in aqueous solutions was also gained from experiments performed at acidic, neutral and alkali pH's. Moreover, biochemical features dealing with the substrate specificity of several bacterial and yeast base excision repair enzymes to remove isodialuric acid from site-specifically modified DNA fragments were determined.

DNA Damage↗
PubMed2006

Characterization of lysine-guanine cross-links upon one-electron oxidation of a guanine-containing oligonucleotide in the presence of a trilysine peptide.

Formation of DNA-protein cross-links involving the initial formation of a guanine radical cation was investigated. For this purpose, riboflavin-mediated photosensitization of a TGT oligonucleotide in aerated aqueous solution in the presence of the KKK tripeptide was performed. We have shown that the nucleophilic addition of the epsilon-amino group of the central lysine residue of KKK to the C8 atom of either the guanine radical cation or its deprotonated form gives rise to the efficient formation of a Nepsilon-(guanin-8-yl)-lysine cross-link. Interestingly, the time course of formation of the above-mentioned cross-link was found to be not linear with the time of irradiation, and its formation rapidly reached a plateau. This is explained by secondary decomposition of the initially generated cross-link which could be further oxidized more efficiently than starting TGT oligonucleotide. One-electron oxidation of the initially generated cross-link was found to produce mainly two diastereomeric cross-links exhibiting a spiroimino-trilysine-dihydantoin structure as inferred from enzymatic digestion, CD, UV, NMR and mass spectrometry measurements. In addition, other minor cross-links, for which formation was favored at acidic pH, were assigned as lysine-guanine adducts in which the modified guanine base exhibits a guanidino-trilysine-iminohydantoin structure. A proposed mechanism for the formation of the different detected oligonucleotide-peptide cross-links is given. The high yield of formation of the detected cross-links strongly suggests that a DNA-protein cross-link involving a lysine residue linked to the C8 position of guanine could be generated in cellular systems if a lysine is located in the close vicinity of a guanine radical cation.

Chromatography, High Pressure Liquid↗
PubMed2006

Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs.

For many years, Taq polymerase has served as the stalwart enzyme in the PCR amplification of DNA. However, a major limitation of Taq is its inability to amplify damaged DNA, thereby restricting its usefulness in forensic applications. In contrast, Y-family DNA polymerases, such as Dpo4 from Sulfolobus solfataricus, can traverse a wide variety of DNA lesions. Here, we report the identification and characterization of five novel thermostable Dpo4-like enzymes from Acidianus infernus, Sulfolobus shibatae, Sulfolobus tengchongensis, Stygiolobus azoricus and Sulfurisphaera ohwakuensis, as well as two recombinant chimeras that have enhanced enzymatic properties compared with the naturally occurring polymerases. The Dpo4-like polymerases are moderately processive, can substitute for Taq in PCR and can bypass DNA lesions that normally block Taq. Such properties make the Dpo4-like enzymes ideally suited for the PCR amplification of damaged DNA samples. Indeed, by using a blend of Taq and Dpo4-like enzymes, we obtained a PCR amplicon from ultraviolet-irradiated DNA that was largely unamplifyable with Taq alone. The inclusion of thermostable Dpo4-like polymerases in PCRs, therefore, augments the recovery and analysis of lesion-containing DNA samples, such as those commonly found in forensic or ancient DNA molecular applications.

Archaea↗
PubMed2006

Nuclear magnetic resonance studies of the 4R and 4S diastereomers of spiroiminodihydantoin 2'-deoxyribonucleosides: absolute configuration and conformational features.

The present study was aimed at gaining further insights into stereochemical and conformational features of the 4R and 4S diastereomers of spiroiminodihydantoin 2'-deoxyribonucleosides that have been shown to be the predominant singlet oxygen oxidation products of 2'-deoxyguanosine in aqueous solutions. It may be added that spiroiminodihydantoin derivatives are efficiently generated by one-electron and singlet oxygen oxidation of the 8-oxo-7,8-dihydroguanine moiety of several nucleic acid components including nucleosides, nucleotides, and oligonucleotides. The reported structural data on the pair of diastereomeric spiroiminodihydantoin 2'-deoxyribonucleosides 1 and 2 are mostly inferred from extensive (1)H and (13)C NMR analyses including two-dimensional nuclear Overhauser effect measurements performed in both D(2)O and dimethyl sulfoxide. This approach that has been shown previously to be suitable to assign the stereochemistry of the base moiety of oxidized pyrimidine nucleosides was completed by molecular modeling and quantum mechanics studies. Thus, application of these two complementary approaches together with the consideration of the results of a recent relevant quantum mechanic study has allowed the assignment of the absolute stereoconfiguration of the C-4 carbon of diastereomers 1 and 2. In addition, information is provided on the conformational features of the 2-deoxyribose moiety and the orientation of the base around the N-glycosidic bond of both 2'-deoxyribonucleosides 1 and 2.

Carbon↗
PubMed2006

Minor contribution of direct ionization to DNA base damage inducedby heavy ions.

PURPOSE: The deleterious processes triggered by heavy ions on DNA were studied through the determination of the yield of a series of oxidized bases. Emphasis was placed on the estimation of the respective contribution of direct ionization and indirect effects, mostly by comparison with low linear energy transfer (LET) gamma-rays. MATERIAL AND METHODS: DNA samples and human monocytes were exposed either to gamma-rays emitted by a (60)Co source or to (12)C(6+) or (36)Ar(18+) ions. The levels of thymidine and 2'-deoxyguanosine oxidation products were determined by liquid chromatography coupled to tandem mass spectrometry subsequently to DNA digestion into nucleosides. RESULTS: The yields of thymidine lesions were similar to those of 8-oxo-7,8-dihydro-2'-deoxyguanosine within isolated DNA exposed either to gamma-rays or argon ions. Addition of spermine and Tris aimed at minimizing the indirect effect modified this ratio to the same extent with both types of radiation. In cells, the level of radiation-induced base damage was found to be correlated with the radiolytic yield of degrees OH that depends on the LET of the particle. In addition, radiation-induced thymidine and 2'-deoxyguanosine lesions were produced in similar amounts. In contrast, oxidation of 2'-deoxyguanosine was the main process when ionization was triggered in cellular DNA by ultraviolet laser-induced biphotonic processes. CONCLUSIONS: Predominant oxidation of 2'-deoxyguanosine is expected to be the hallmark of direct DNA ionization. The observation that thymidine and 2'-deoxyguanosine are equally damaged rules out a major contribution of the direct ionization in radiation-induced base damage to both isolated and cellular DNA by heavy ions. Dependence of the yield of lesions on the LET provides further support for this conclusion.

DNA↗
PubMed2005

Proceedings of a workshop on DNA adducts: biological significance and applications to risk assessment Washington, DC, April 13-14, 2004.

In April 2004, the Health and Environmental Sciences Institute, a branch of the International Life Sciences Institute, with support from the National Institute of Environmental Health Sciences, organized a workshop to discuss the biological significance of DNA adducts. Workshop speakers and attendees included leading international experts from government, academia, and industry in the field of adduct detection and interpretation. The workshop initially examined the relationship between measured adduct levels in the context of exposure and dose. This was followed by a discussion on the complex response of cells to deal with genotoxic insult in complex, interconnected, and interdependent repair pathways. One of the major objectives of the workshop was to address the recurring question about the mechanistic and toxicological relevance of low-concentration measured adducts and the presentations in the session entitled "Can low levels of DNA adducts predict adverse outcomes?" served as catalysts for further discussions on this subject during the course of the workshop. Speakers representing the regulatory community and industry reviewed the value, current practices, and limitations of utilizing DNA adduct data in risk assessment and addressed a number of practical questions pertaining to these issues. While no consensus statement emerged on the biological significance of low levels of DNA adducts, the workshop concluded by identifying the need for more experimental data to address this important question. One of the recommendations stemming from this workshop was the need to develop an interim "decision-logic" or framework to guide the integration of DNA adduct data in the risk assessment process. HESI has recently formed a subcommittee consisting of experts in the field and other key stakeholders to address this recommendation as well as to identify specific research projects that could help advance the understanding of the biological significance of low levels of DNA adducts.

Animals↗
PubMed2005

Repair of the three main types of bipyrimidine DNA photoproducts in human keratinocytes exposed to UVB and UVA radiations.

Induction of DNA damage by solar UV radiation is a key event in the development of skin cancers. Bipyrimidine photoproducts, including cyclobutane pyrimidine dimers (CPDs), (6-4) photoproducts (64 PPs) and their Dewar valence isomers, have been identified as major UV-induced DNA lesions. In order to identify the predominant and most persistent lesions, we studied the repair of the three types of photolesions in primary cultures of human keratinocytes. Specific and quantitative data were obtained using HPLC associated with tandem mass spectrometry. As shown in other cell types, 64 PPs are removed from UVB-irradiated keratinocytes much more efficiently than CPDs. In contrast, CPDs are still present in high amounts when cells recover their proliferation capacities after cell cycle arrest and elimination of a part of the population by apoptosis. The predominance of CPDs is still maintained when keratinocytes are exposed to a combination of UVB and UVA. Under these conditions, 64 PPs are converted into their Dewar valence isomers that are as efficiently repaired as their (6-4) precursors. Exposure of cells to pure UVA radiation generates thymine cyclobutane dimers that are slightly less efficiently repaired than CPDs produced upon UVB irradiation. Altogether, our results show that CPDs are the most frequent and the less efficiently repaired bipyrimidine photoproducts irrespectively of the applied UV treatment.

Apoptosis↗
PubMed2005

Identification of the main oxidation products of 8-methoxy-2'-deoxyguanosine by singlet molecular oxygen.

It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides.

Chromatography, High Pressure Liquid↗
PubMed2005

Detection of chlorinated DNA and RNA nucleosides by HPLC coupled to tandem mass spectrometry as potential biomarkers of inflammation.

Upon inflammation, activated neutrophils secrete myeloperoxidase, an enzyme able to generate hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. An analytical method, involving HPLC coupled to electrospray tandem mass spectrometry, has been set-up to detect low levels of HOCl-induced nucleic acids lesions, including both ribo and 2'-deoxyribonucleoside derivatives of 8-chloroguanine, 8-chloroadenine and 5-chlorocytosine. Validation of the developed method was achieved using isolated cells treated with HOCl. The method was found to be sensitive enough to allow the measurement of background levels of 5-chloro-2'-deoxycytidine in the DNA of human white blood cells isolated from 7 mL of blood.

Adenine↗
PubMed2005

Ultraviolet radiation-mediated damage to cellular DNA.

Emphasis is placed in this review article on recent aspects of the photochemistry of cellular DNA in which both the UVB and UVA components of solar radiation are implicated individually or synergistically. Interestingly, further mechanistic insights into the UV-induced formation of DNA photoproducts were gained from the application of new accurate and sensitive chromatographic and enzymic assays aimed at measuring base damage. Thus, each of the twelve possible dimeric photoproducts that are produced at the four main bipyrimidine sites can now be singled out as dinucleoside monophosphates that are enzymatically released from UV-irradiated DNA. This was achieved using a recently developed high-performance liquid chromatography-tandem mass spectrometry assay (HPLC-MS/MS) assay after DNA extraction and appropriate enzymic digestion. Interestingly, a similar photoproduct distribution pattern is observed in both isolated and cellular DNA upon exposure to low doses of either UVC or UVB radiation. This applies more specifically to the DNA of rodent and human cells, the cis-syn cyclobutadithymine being predominant over the two other main photolesions, namely thymine-cytosine pyrimidine (6-4) pyrimidone adduct and the related cyclobutyl dimer. UVA-irradiation was found to generate cyclobutane dimers at TT and to a lower extent at TC sites as a likely result of energy transfer mechanism involving still unknown photoexcited chromophore(s). Oxidative damage to DNA is also induced although less efficiently by UVA-mediated photosensitization processes that mostly involved 1O2 together with a smaller contribution of hydroxyl radical-mediated reactions through initially generated superoxide radicals.

Animals↗
PubMed2005

Repair of oxidative damage of thymine by HeLa whole-cell extracts: simultaneous analysis using a microsupport and comparison with traditional PAGE analysis.

In mammalian cells, the base excision repair (BER) pathway allows the remove of small DNA base lesions such as oxidized bases. It is initiated by glycosylases that removed the modified base leaving an abasic site that is subsequently processed by AP endonuclease activities. Measurement of BER activities in cell extracts is time consuming and hazardous when radioactive material is used. We report in this study, the parallelized fluorescent analysis of excision of several oxidation products of thymine by cell extracts. To conduct the study, 5-(hydroxymethyl)uracil, 5-formyluracil, 5-carboxyuracil and formylamine together with uracil and the control thymine, were incorporated into oligonucleotides of identical sequences and paired either with adenine or with guanine containing DNA fragments. The oligonucleotides were fixed by sandwich hybridization in wells of a microplate (OLISA technology). Excision by HeLa extracts of the six different DNA base lesions could be followed simultaneously in the same well. Our results showed that the extent of excision of the lesions was the same on support and in solution using classical PAGE analysis approach with modified (32)P-labeled oligonucleotides. We demonstrated that the simultaneous analysis on support is a successful approach to facilitate high-throughput screening of BER activities present in cell extracts. Moreover, extended study of 5-carboxyuracil revealed that this lesion displays similar biological properties as 5-formyluracil.

Cell-Free System↗
PubMed2005

Enhancement of the in vitro transcription by T7 RNA polymerase of short DNA templates containing oxidative thymine lesions.

5-(Hydroxymethyl)uracil, 5-formyluracil or 5-carboxyuracil can be formed in DNA upon exposure to ionizing radiations. We report the effect of the latter oxidative lesions on the in vitro transcription by T7 RNA polymerase using a single-stranded short model template. Kinetic experiments showed that the transcription yield was increased with the modified template. Due to the unforeseen presence of a hairpin structure within the transcribed RNA, we postulated that we are in the presence of a T7 RNA polymerase pausing signal and that the lesions within this critical DNA structure could interfere with normal regulation of RNA transcription.

Bacteriophage T7↗
PubMed2005

Synthesis of a convenient thymidine glycol phosphoramidite monomer and its site-specific incorporation into DNA fragments.

An original phosphoramidite building block of the thymidine glycol lesion has been prepared taking into account the additional diol function and the high lability of this oxidatively induced nucleobase damage. Then the modified nucleoside was site-specifically inserted into DNA fragments by solid support assembling followed by a "one-step" mild final deprotection treatment.

DNA Damage↗
PubMed2004

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells.

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.

8-Hydroxy-2'-Deoxyguanosine↗