المؤلفون: Denis Biard, Alexandre T. Vessoni, Annabel Quinet, Alain Sarasin, Davi Jardim Martins, Carlos Frederico Martins Menck, Anne Stary

المساهمون: Universidade de São Paulo = University of São Paulo (USP), Institut de Radiobiologie Cellulaire et Moléculaire (IRCM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Intégrité du génome et cancers (IGC), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut Gustave Roussy (IGR)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), University of São Paulo (USP), Stabilité Génétique et Oncogenèse (UMR 8200), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS)

المصدر: Nucleic Acids Research
Nucleic Acids Research, 2016, 44 (12), pp.5717-5731. ⟨10.1093/nar/gkw280⟩
Nucleic Acids Research, Oxford University Press, 2016, 44 (12), pp.5717-5731. ⟨10.1093/nar/gkw280⟩

مصطلحات موضوعية: DNA Replication, 0301 basic medicine, MESH: Human Cells, DNA Repair, Ultraviolet Rays, DNA repair, DNA polymerase, DNA damage, MESH: DNA Lesion, Genetic Vectors, DNA, Single-Stranded, Pyrimidine dimer, [SDV.CAN]Life Sciences [q-bio]/Cancer, DNA-Directed DNA Polymerase, Genome Integrity, Repair and Replication, Adenoviridae, S Phase, 03 medical and health sciences, Transduction, Genetic, Genetics, Postreplication repair, Humans, Cell Line, Transformed, MESH: Xeroderma Pigmentosum, MESH: DNA Damage, MESH: Translesion Synthesis, biology, Genome, Human, DNA replication, Nuclear Proteins, MESH: Ultraviolet, Fibroblasts, DNA Replication Fork, Nucleotidyltransferases, Cell biology, DNA-Binding Proteins, 030104 developmental biology, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Pyrimidine Dimers, biology.protein, REV1, MESH: Coronary Disease, Deoxyribodipyrimidine Photo-Lyase, DNA Damage

الوصف: International audience; Ultraviolet-induced 6-4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD) can be tolerated by translesion DNA polymerases (TLS Pols) at stalled replication forks or by gap-filling. Here, we investigated the involvement of Pol, Rev1 and Rev3L (Pol catalytic subunit) in the specific bypass of 6-4PP and CPD in repair-deficient XP-C human cells. We combined DNA fiber assay and novel methodologies for detection and quantification of singlestranded DNA (ssDNA) gaps on ongoing replication forks and postreplication repair (PRR) tracts in the human genome. We demonstrated that Rev3L, but not Rev1, is required for postreplicative gapfilling, while Pol and Rev1 are responsible for TLS at stalled replication forks. Moreover, specific photolyases were employed to show that in XP-C cells, CPD arrest replication forks, while 6-4PP are responsible for the generation of ssDNA gaps and PRR tracts. On the other hand, in the absence of Pol or Rev1, both types of lesion block replication forks progression. Altogether, the data directly show that, in the human genome, Pol and Rev1 bypass CPD and 6-4PP at replication forks, while only 6-4PP are also tolerated by a Pol-dependent gap-filling mechanism, independent of S phase.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b5b6b3e6c5f04e7774ad3693216e7f7bTest
https://hal.science/hal-03679922/documentTest

المؤلفون: Carlos Frederico Martins Menck, Gustavo P. Amarante-Mendes, James E. Cleaver, Alain Sarasin, Melissa Gava Armelini, Marila Cordeiro-Stone, Anne Stary, Jacqueline F. Jacysyn, Patricia Kannouche, Keronninn M. Lima-Bessa, Vanessa Chiganças

المصدر: DNA Repair. 5:925-934

مصطلحات موضوعية: DNA Replication, DNA, Complementary, Xeroderma pigmentosum, DNA Repair, Ultraviolet Rays, DNA repair, DNA polymerase, DNA polymerase II, Blotting, Western, Genetic Vectors, Green Fluorescent Proteins, DNA-Directed DNA Polymerase, DNA polymerase eta, Biochemistry, Adenoviridae, Transduction, Genetic, medicine, Humans, Molecular Biology, Cells, Cultured, Polymerase, Xeroderma Pigmentosum, biology, Cell Biology, medicine.disease, Molecular biology, Proliferating cell nuclear antigen, biology.protein, Nucleotide excision repair

الوصف: Xeroderma pigmentosum (XP) is an autosomal recessive photosensitive disorder with an extremely high incidence of skin cancers. Seven complementation groups, corresponding to seven proteins involved in nucleotide excision repair (NER), are associated with this syndrome. However, in XP variant patients, the disorder is caused by defects in DNA polymerase eta; this error prone polymerase, encoded by POLH, is involved in translesion DNA synthesis (TLS) on DNA templates damaged by ultraviolet light (UV). We constructed a recombinant adenovirus carrying the human POLH cDNA linked to the EGFP reporter gene (AdXPV-EGFP) and infected skin fibroblasts from both XPV and XPA patients. Twenty-four hours after infection, the DNA polymerase eta-EGFP fusion protein was detected by Western blot analysis, demonstrating successful transduction by the adenoviral vector. Protein expression was accompanied by reduction in the high sensitivity of XPV cells to UV, as determined by cell survival and apoptosis-induction assays. Moreover, the pronounced UV-induced inhibition of DNA synthesis in XPV cells and their arrest in S phase were attenuated in AdXPV-EGFP infected cells, confirming that the transduced polymerase was functional. However, over-expression of polymerase eta mediated by AdXPV-EGFP infection did not result in enhancement of cell survival, prevention of apoptosis, or higher rate of nascent DNA strand growth in irradiated XPA cells. These results suggest that TLS by DNA polymerase eta is not a limiting factor for recovery from cellular responses induced by UV in excision-repair deficient fibroblasts.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::e2b3146642fbf3c8d24d50199b58bcf9Test
https://doi.org/10.1016/j.dnarep.2006.05.037Test

المؤلفون: Alain Sarasin, Anne Stary, S A C Jorge, David H. Phillips, Carlos Frederico Martins Menck, Martin R. Osborne, Helmut Sies

المصدر: DNA Repair. 1:369-378

مصطلحات موضوعية: DNA Replication, GC Rich Sequence, Guanine, Ozone, DNA damage, Genetic Vectors, Molecular Sequence Data, Mutagen, Biology, Kidney, medicine.disease_cause, Biochemistry, chemistry.chemical_compound, Oxidants, Photochemical, medicine, Humans, Molecular Biology, Cells, Cultured, Cell Line, Transformed, chemistry.chemical_classification, Genetics, Reactive oxygen species, Base Sequence, Mutagenicity Tests, DNA, Cell Biology, AT Rich Sequence, Lac Operon, chemistry, 8-Hydroxy-2'-Deoxyguanosine, Mutation, Carcinogenesis, Genotoxicity, DNA Damage

الوصف: Ozone is an important factor in urban pollution and represents a major concern for human health. The chemical reactivity of ozone toward biological targets and particularly its genotoxicity supports a possible link between exposure and cancer risk, but no molecular data exist on its mutagenic potential in human cells. Using a shuttle vector, we showed that ozone is indeed a potent mutagen and we characterized the mutation spectrum it produced in human cells. Almost all mutations are base substitutions, essentially located at G:Cs (75%), typical of reactive oxygen species (ROS), but occurring in a specific pattern, i.e. a similar extent of GC:TA (28%), GC:CG (23%) and GC:AT (23%). The targeted distribution of mutations and identification of hotspot sequences define the first molecular fingerprint of mutations induced by ozone in human cells. Possible applications derived from our results with respect to ozone genotoxicity should help determining quantifiable biomarkers of ozone exposure in human health, especially for carcinogenesis.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::30f9110631787e44dbf701c7f938dbd2Test
https://doi.org/10.1016/s1568-7864Test(02)00011-3

المؤلفون: Simone Benhamou, Alain Sarasin, Claire Marionnet, Annie Benoit, Anne Stary

المصدر: Journal of Molecular Biology. 252:550-562

مصطلحات موضوعية: Xeroderma pigmentosum, DNA Repair, Cell Survival, Ultraviolet Rays, DNA repair, Genetic Vectors, Molecular Sequence Data, Trichothiodystrophy, Biology, medicine.disease_cause, Cell Line, Structural Biology, medicine, Humans, Point Mutation, Amino Acid Sequence, Mutation frequency, Molecular Biology, Cell Line, Transformed, Xeroderma Pigmentosum, Mutation, Mutation Spectra, Base Sequence, Point mutation, Fibroblasts, medicine.disease, Molecular biology, Lac Operon, Mutagenesis, Hair Diseases, Carcinogenesis

الوصف: To study the relationships between mutagenesis and carcinogenesis, we compared the mutations and their frequency induced by ultraviolet irradiation at 254 nm (UV-C) in XP-D (GM-08207B/XP6BE), TTD/XP-D (TTD1VI-LAS-KMT11) and wild-type (MRC-5V1) human cells. XP-D and TTD/XP-D cells, mutated in the same XP-D/ERCC2 gene, are deficient in nucleotide excision repair. Whereas XP-D patients develop early skin tumors, TTD patients do not exhibit abnormal levels of cancers. After verification of UV hypersensitivity and DNA repair defect of the immortalized cell lines XP-D and TTD compared with a wild-type cell line, UV-induced mutagenesis was studied with a new shuttle vector pR2, carrying the target lacZ ′ gene. The UV-mutation frequencies in XP-D and TTD cells were similar and significantly increased compared with normal cells. Sequence analysis of 312 independent mutant plasmids revealed that more rearrangements were induced in TTD cells (16%) than in XP-D (5%) and normal cells (1%), while XP-D cells exhibited a twofold higher rate of tandem mutations compared with TTD and normal cells. In the three cell lines, a predominance of G:C to A:T transitions was found, especially in XP-D cells (87%) and most mutations were targeted on dipyrimidine sites, chiefly on the cytosine at 5′-TC-3′ sites. The types of UV-induced point mutations in TTD cells were, however, more similar to those in normal cells than those found in XP-D cells. XP-D mutations were preferentially located in 5′-TCPur3′ sites, while mutations in normal and TTD cells were mostly at 5′-TCC-3′ sites. Analysis of mutation spectra revealed differences in the location of the mutational hotspots between the three lines. Although the mutation frequency of the UV-irradiated pR2 vector is much higher in TTD and XP-D cells than in normal cells, the mutation spectrum is closer between TTD and normal cells as compared with XP-D cells. These dissimilarities could contribute to an explanation of some of the differences between the two syndromes.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::30ac6b2d45181a14899a48cbde461d81Test
https://doi.org/10.1006/jmbi.1995.0519Test

المؤلفون: Alain Sarasin, Carlos Frederico Martins Menck, Anne Stary

المصدر: Mutation Research/Environmental Mutagenesis and Related Subjects. 272:101-110

مصطلحات موضوعية: Herpesvirus 4, Human, Methylnitronitrosoguanidine, Base Sequence, Mutagenicity Tests, Base pair, Point mutation, Genetic Vectors, Molecular Sequence Data, lac operon, Locus (genetics), DNA, Simian virus 40, Biology, beta-Galactosidase, Toxicology, Molecular biology, Cell Line, Plasmid, Shuttle vector, Genetics, Humans, Low copy number, Gene, Plasmids

الوصف: In order to analyze the mechanisms of mutagenesis in human cells, we have established a human 293 cell-derived line containing a permanent mutagenesis target, the bacterial lacZ' gene, on an episomal EBV/SV40-based shuttle vector. This plasmid was maintained at a low copy number per cell which rendered it closer to an endogenous gene as compared to the usual transient shuttle vectors. Transient amplification of vectors, inside the host cell due to expression of the SV40 T-antigen, allowed the recovery of a large number of bacterial colonies transformed by plasmids extracted from human cells. Mutations produced in human host cells on the lacZ' locus were easily and rapidly scored and identified in bacteria using the blue/white color assay. Over a 6-month period in culture, we have shown that the lacZ' gene exhibited a low background frequency of point mutations (4.8 x 10(-6)). The efficiency of our system for detecting genotoxic-induced mutations was investigated by treating cells with a potent mutagen, the direct alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A significant increase (230-fold) in the frequency of single-base substitutions was observed after MNNG treatment. In total, 63 MNNG-induced independent mutations were characterized. All substitutions but one involved G:C base pairs with 89% being G:C to A:T transitions which is consistent with the MNNG mutagenic specificity already reported in bacteria and mammalian cells. Mutations were distributed along the two strands of the lacZ' gene and there was no obvious influence of either the 5' or the 3' flanking base near the G:C to A:T transition sites. The low spontaneous point mutation frequency on the mutagenesis locus and the ability to detect induced point mutations indicate that this system could be readily used in human mutagenesis studies at the molecular level.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a023a19f3dbc78320ba754723f6f4ecfTest
https://doi.org/10.1016/0165-1161Test(92)90038-n

المؤلفون: Alain Sarasin, Anne Stary

المصدر: Journal of General Virology. 73:1679-1685

مصطلحات موضوعية: Herpesvirus 4, Human, Antigens, Polyomavirus Transforming, Genetic Vectors, Molecular Sequence Data, Population, Simian virus 40, Biology, Origin of replication, DNA sequencing, Virus, chemistry.chemical_compound, Plasmid, Shuttle vector, Virology, Humans, education, Gene Rearrangement, Recombination, Genetic, education.field_of_study, Base Sequence, Gene Amplification, Gene rearrangement, Molecular biology, chemistry, Hybridization, Genetic, DNA, HeLa Cells, Plasmids

الوصف: We analysed the DNA rearrangements that occurred during the integration and amplification of an Epstein-Barr virus (EBV)-simian virus 40 (SV40) hybrid shuttle vector in human cells. The human HeLa cell line was episomally transformed with the EBV-SV40 p205-GTI plasmid. After a 2 month culture in a selective medium, a HeLa cell-derived population (H-G1 cells) was obtained in which the p205-GTI vector was integrated as a single intact copy deleted in the EBV latent origin of replication (OriP). Sequencing data showed that the endpoints of the plasmid sequences, at the plasmid-cell DNA junctions, are located within the two essential elements of EBV OriP, which may form several secondary structures. This result suggests that a specific DNA sequence (OriP) or palindromic structures could play a role in this integration process. This represents the first fully characterized site of integration of an EBV vector in human cells. The transient expression of the SV40 large T antigen in H-G1 cells leads to the appearance of episomal molecules with an extremely heterogeneous size pattern. Individual analysis of these episomes after rescue in bacteria indicated that they retained sequences of both the p205-GTI plasmid and cellular DNA. Comparison of the structure of these circular DNAs with those of the integrated p205-GTI copy indicated that large T antigen expression in human cells leads to the amplification of the integrated shuttle vector according to the ‘onion skin’ model developed for transformed rodent cells. Indeed, amplified sequences were collinear with the integrated p205-GTI copy and its surrounding cellular sequences, distributed almost equally around the SV40 replication origin, and circularized by illegitimate recombination which did not involve specific nucleotide sequences. This system is of interest in that it enables easy recovery of individual recombined molecules in host bacteria. Each isolated clone contains a unique recombination junction which is easily and rapidly characterized and sequenced.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::517d9800b0b4eae3f5488672ee78aa37Test
https://doi.org/10.1099/0022-1317-73-7-1679Test

المؤلفون: Michael R. James, André Cordier, Alain Sarasin, Denis S.F. Biard

المصدر: Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1130:68-74

مصطلحات موضوعية: Chloramphenicol O-Acetyltransferase, Isopropyl Thiogalactoside, Herpesvirus 4, Human, Transcription, Genetic, Operon, Genetic Vectors, Molecular Sequence Data, Biophysics, Repressor, lac operon, Biology, Lac repressor, Biochemistry, Cell Line, Structural Biology, Gene expression, Escherichia coli, Genetics, Humans, Promoter Regions, Genetic, Regulation of gene expression, Reporter gene, Base Sequence, Promoter, beta-Galactosidase, Molecular biology, Repressor Proteins, carbohydrates (lipids), Zinc, Gene Expression Regulation, Lac Operon, Cadmium, Plasmids

الوصف: We have investigated the use of various Epstein-Barr virus (EBV)-based vectors bearing the two components of the Escherichia coli lac operator-repressor (lacO, lacI) complex. Our aim was to develop a model system of gene expression by looking at the transcription of the bacterial beta-galactosidase coding gene (lacZ) in 293 human embryonic kidney cells. Several vectors have been built carrying different promoters upstream of the lacI and lacZ genes and in which natural or synthetic operator sequences were inserted in the 5' part of the lacZ gene. In transient expression assays we achieved efficient lacZ gene repression which could be released by the specific inducer isopropyl beta-D-thiogalactoside (IPTG). A stable transformed cell line carrying two EBV-derived plasmids with the building blocks of the lac operator/repressor system was established. This cell line allowed us to achieve a wide range of lacZ gene regulation. In this cell line IPTG alone could remove the repression to trigger a 5-fold increase of lacZ expression. Heavy metal ions, which induced the mouse metallothionein I promoter located upstream of the lacZ gene, added together with IPTG gave rise to a 40-fold induction of lacZ expression.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ca8db58cfc847624ecbd7ecef6088526Test
https://doi.org/10.1016/0167-4781Test(92)90463-a

المؤلفون: Anne Stary, Alain Sarasin

المصدر: Nucleic Acids Research. 20:4269-4274

مصطلحات موضوعية: DNA Replication, Recombination, Genetic, Genetics, Base Sequence, Antigens, Polyomavirus Transforming, FLP-FRT recombination, Genetic Vectors, Molecular Sequence Data, Alu element, Simian virus 40, Biology, Origin of replication, chemistry.chemical_compound, Plasmid, Proviruses, chemistry, Humans, Ectopic recombination, In vitro recombination, Recombination, DNA, HeLa Cells, Plasmids, Repetitive Sequences, Nucleic Acid

الوصف: In a human HeLa derived-cell line carrying permanently a single integrated copy of an SV40 shuttle vector, the transient expression of the SV40 T-antigen led to the production of heterogeneous populations of circular DNA molecules which retained both integrated vector and its surrounding cellular sequences. Comparison between the integrated copy and the linear maps of 80 different plasmids rescued in bacteria suggested that the formation of circular DNA was the result of bidirectional replication from the SV40 origin of replication followed by a single intramolecular joining leading to the cyclization of the replicated molecules. Sequence analysis of 45 recombinational junctions demonstrated that the cyclization occurred via illegitimate recombination process which did not require preferential nucleotide sequence at the joining sites. However, extensive characterization of recombination junctions revealed that the sequences involved in the recombination at each side of the SV40 origin of replication were not randomly distributed, suggesting the presence of regions which were more prone to be involved in the illegitimate recombination process in human cells. Search of common features usually implied in illegitimate recombination in mammalian cells revealed some association of these regions with palindromes, A + T-rich DNA segments, alternating purine/pyrimidine sequences and Alu family repeats.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b54f6b011193081c9f5d565a9058426dTest
https://doi.org/10.1093/nar/20.16.4269Test

المؤلفون: Alain Sarasin, G. Renault, A. Margot, Alain Gentil, R. Teoule

المصدر: Nucleic Acids Research. 18:6361-6367

مصطلحات موضوعية: DNA Replication, Pyrimidine, Guanine, Genetic Vectors, Molecular Sequence Data, Restriction Mapping, Simian virus 40, Biology, Transfection, Cell Line, chemistry.chemical_compound, Genetics, Activation-induced (cytidine) deaminase, Animals, Uracil, Base Sequence, Nucleic Acid Heteroduplexes, Molecular biology, Thymine, Mutagenesis, Insertional, Oligodeoxyribonucleotides, chemistry, DNA glycosylase, DNA, Viral, biology.protein, Indicators and Reagents, Uracil nucleotide, Cytosine

الوصف: The processing of a unique uracil in DNA has been studied in mammalian cells. A synthetic oligodeoxyribonucleotide carrying a potential Bgl II restriction site, where one base has been substituted with a uracil, was inserted in the early intron of SV40 genome. Various heteroduplexes were constructed in such a manner that the restitution of an active Bgl II restriction site corresponds in each case to the specific substitution of the uracil by one of the four bases normally present in the DNA. DNA cuts by this restriction enzyme in one or several constructed heteroduplexes immediately determine the type of base pair substitution produced at the site of the U residue. When the uracil is inserted opposite a purine it is fully repaired; when facing a guanine it is replaced by a cytosine and opposite an adenine it is replaced by a thymine. These results indicate the error-free repair of uracil when it appears in the cell with the usual mechanisms such as cytosine deamination or incorporation of dUTP in place of dTTP during replication. When the uracil is inserted opposite a pyrimidine no error free repair at all is detected for U:C or U:T mismatches. It appears, moreover, that in approximately 18% of the cases U:T mismatch leads to a C:G base pairing. In the majority of the U:pyrimidine mismatches, mutations occur in the vicinity of the uracil, including base substitutions and frameshifts by addition of one or several bases.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::91e75c0e3303bf7883a521ed41a4de79Test
https://doi.org/10.1093/nar/18.21.6361Test

المؤلفون: M.R. James, Alain Sarasin, Carlos Frederico Martins Menck, A. Benoit

المصدر: Scopus-Elsevier

مصطلحات موضوعية: DNA Replication, viruses, Genetic Vectors, Restriction Mapping, Deoxyribonuclease HindIII, Simian virus 40, Biology, Transfection, Virus Replication, Origin of replication, Genome, Virus, Cell Line, chemistry.chemical_compound, Capsid, Plasmid, Shuttle vector, Virology, Animals, Electrophoresis, Agar Gel, Gene Rearrangement, Genetics, Virion, Blotting, Southern, chemistry, Lytic cycle, DNA, Viral, DNA, Plasmids

الوصف: Simian virus 40 (SV40)-based shuttle vectors, containing the SV40 late genes, can be packaged as infectious pseudovirions. In terms of their function as bacterial plasmids, modifications in the overall size of these plasmids can be tolerated within a very wide range, which has allowed us to determine the requirements for SV40 encapsidation, free of the more stringent limitations of SV40 virus. Monkey COS7 cells were transfected with over- and undersized SV40-based shuttle virus plasmids and their progeny have been analysed to follow the stability and evolution of these genomes. Two of the three plasmids analysed undergo recombination, generating molecules with sizes of between 4.0 and to 4.8 kb which were selected after multiple lytic cycles. This size range may correspond to the DNA lengths preferentially packaged in SV40 capsids. The structure of the rearranged plasmids indicates that there is a strong selective pressure for genomes that retain the functions necessary for replication and virus production. Depending on the parent DNA, two main classes of rearrangements were generated: duplications in tandem with the SV40 origin of replication and deletions. Both classes are probably a result of selective size and replicative advantages, which are then biologically amplified during plasmid transmission as virus particles.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5f1cf685d4903649f8da415a33def881Test
https://doi.org/10.1099/0022-1317-71-1-143Test