(5′S)-8,5′-Cyclo-2′-deoxyguanosine Is a Strong Block to Replication, a Potent pol V-Dependent Mutagenic Lesion, and Is Inefficiently Repaired in Escherichia coli

8,5′-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5′S)-8,5′-cyclo-2′-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was \u3c1% in the wild-type strain, which increased to 5.5% with SOS. Viability decreased further in a pol II- strain, whereas it increased considerably in a pol IV- strain. Remarkably, no progeny was recovered from a pol V- strain, indicating that pol V is absolutely required for bypassing S-cdG. Progeny analyses indicated that S-cdG is significantly mutagenic, inducing ∼34% mutation with SOS. Most mutations were S-cdG → A mutations, though S-cdG → T mutation and deletion of 5′C also occurred. Incisions of purified UvrABC nuclease on S-cdG, S-cdA, and C8-dG-AP on a duplex 51-mer showed that the incision rates are C8-dG-AP \u3e S-cdA \u3e S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli

Structures of (5′S)-8,5′-Cyclo-2′-deoxyguanosine Mismatched with dA or dT

pubs.acs.org/cr

Unexpected Role for Helicobacter pylori DNA Polymerase I As a Source of Genetic Variability

Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5′- 3′ exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity

Synthesis of the spore photoproduct and structural analogs

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2D Nanostructures based on DNA-Porphyrin Hybrids

International audienceThe use of DNA as support of information for the fabrication of supramolecular nanostructures has been extensively reported over the last decade. Different strategies were used for this purposes either based purely on oligonucleotides or by combining oligonucleotides with organic molecules. On the first hand, the approach based on pure oligonucleotide can be achieved using double crossover DNA strands 1 or via DNA origami. 2 The second strategy relies on the use of small organic molecules to define the angle in the nanostructures. 3 These organic molecules can be introduced in single stranded DNA (ssDNA) during its synthesis or functionalized afterward with functional DNA. Recently, we reported the synthesis of porphyrin/DNA hybrids by copper-catalyzed Huisgen cycloaddition reaction (CuAAC). We synthesized four DNA/porphyrin derivatives (from the mono to the tetra adduct) containing a 14-mer single stranded DNA. 4 We tested the association of the tetra-adducts with gold nanoparticles, we proved this association but we realized that the formation of double stranded DNA using 14-mer oligonucleotides was not efficient enough under our hydridization conditions. In order to improve the stability and give rise to the formation of 2D nanostructure, we increased the number of bases of our ODN from 14 to 20. Here, we report on the synthesis of two porphyrin/oligonucleotide polyadducts via CuAAC containing two complementary strands (ODN-1: 5'-GCA TTG GTA ACT GTC AGA CCU-3' and ODN-2: 5'-GGT CTG ACA GTT ACC AAT GCU-3') named respectively Pn (n = 1-4) and Pnc (n = 1-4). The hybrids were characterized by absorption spectroscopy and gel electrophoresis. The two complementary hybrids P4 and P4c were associated to form a 2D nanostructure and studied by atomic force microscopy

Excision of oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5methylhydantoin pyrimidine lesion by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

International audienceBackground: (5R⁎) and (5S⁎) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.Methods: Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.Results: In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the kcat/Km ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.Conclusions: The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.General significanceThe study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions

2D Nanostructures based on DNA-Porphyrin Hybrids

International audienceThe use of DNA as support of information for the fabrication of supramolecular nanostructures has been extensively reported over the last decade. Different strategies were used for this purposes either based purely on oligonucleotides or by combining oligonucleotides with organic molecules. On the first hand, the approach based on pure oligonucleotide can be achieved using double crossover DNA strands 1 or via DNA origami. 2 The second strategy relies on the use of small organic molecules to define the angle in the nanostructures. 3 These organic molecules can be introduced in single stranded DNA (ssDNA) during its synthesis or functionalized afterward with functional DNA. Recently, we reported the synthesis of porphyrin/DNA hybrids by copper-catalyzed Huisgen cycloaddition reaction (CuAAC). We synthesized four DNA/porphyrin derivatives (from the mono to the tetra adduct) containing a 14-mer single stranded DNA. 4 We tested the association of the tetra-adducts with gold nanoparticles, we proved this association but we realized that the formation of double stranded DNA using 14-mer oligonucleotides was not efficient enough under our hydridization conditions. In order to improve the stability and give rise to the formation of 2D nanostructure, we increased the number of bases of our ODN from 14 to 20. Here, we report on the synthesis of two porphyrin/oligonucleotide polyadducts via CuAAC containing two complementary strands (ODN-1: 5'-GCA TTG GTA ACT GTC AGA CCU-3' and ODN-2: 5'-GGT CTG ACA GTT ACC AAT GCU-3') named respectively Pn (n = 1-4) and Pnc (n = 1-4). The hybrids were characterized by absorption spectroscopy and gel electrophoresis. The two complementary hybrids P4 and P4c were associated to form a 2D nanostructure and studied by atomic force microscopy

Boronic acid-based fluorescent receptors for selective recognition of thymine glycol.

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