Artifacts associated with the measurement of oxidized DNA bases.

In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, the situation is confusing because variations that may be as large as two orders of magnitude have been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings clearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and -32P--postlabeling may lead to a significant overestimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 10(4) normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7, 8-dihydroadenine,5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolysate prior to the derivatization reaction prevents artefactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2;-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropriate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cell gel electrophoresis (comet) assay

TtcA a new tRNA-thioltransferase with an Fe-S cluster

International audienceTtcA catalyzes the post-transcriptional thiolation of cytosine 32 in some tRNAs. The enzyme from Es-cherichia coli was homologously overexpressed in E. coli. The purified enzyme is a dimer containing an iron–sulfur cluster and displays activity in in vitro assays. The type and properties of the cluster were investigated using a combination of UV-visible absorption , EPR and M ¨ ossbauer spectroscopy, as well as by site-directed mutagenesis. These studies demonstrated that the TtcA enzyme contains a redox-active and oxygen-sensitive [4Fe-4S] cluster, chelated by only three cysteine residues and absolutely essential for activity. TtcA is unique tRNA-thiolating enzyme using an iron–sulfur cluster for catalyzing a non-redox reaction

Undetectable levels of N6-methyl adenine in mouse DNA: Cloning and analysis of PRED28, a gene coding for a putative mammalian DNA adenine methyltransferase.

International audienceThree methylated bases, 5-methylcytosine, N4-methylcytosine and N6-methyladenine (m6A), can be found in DNA. However, to date, only 5-methylcytosine has been detected in mammalian genomes. To reinvestigate the presence of m6A in mammalian DNA, we used a highly sensitive method capable of detecting one N6-methyldeoxyadenosine per million nucleosides. Our results suggest that the total mouse genome contains, if any, less than 10(3) m6A. Experiments were next performed on PRED28, a putative mammalian N6-DNA methyltransferase. The murine PRED28 encodes two alternatively spliced RNA. However, although recombinant PRED28 proteins are found in the nucleus, no evidence for an adenine-methyltransferase activity was detected

Hematopoietic gene promoters subjected to a group-combinatorial study of DNA samples: identification of a megakaryocytic selective DNA signature

Identification of common sub-sequences for a group of functionally related DNA sequences can shed light on the role of such elements in cell-specific gene expression. In the megakaryocytic lineage, no one single unique transcription factor was described as linage specific, raising the possibility that a cluster of gene promoter sequences presents a unique signature. Here, the megakaryocytic gene promoter group, which consists of both human and mouse 5′ non-coding regions, served as a case study. A methodology for group-combinatorial search has been implemented as a customized software platform. It extracts the longest common sequences for a group of related DNA sequences and allows for single gaps of varying length, as well as double- and multiple-gap sequences. The results point to common DNA sequences in a group of genes that is selectively expressed in megakaryocytes, and which does not appear in a large group of control, random and specific sequences. This suggests a role for a combination of these sequences in cell-specific gene expression in the megakaryocytic lineage. The data also point to an intrinsic cross-species difference in the organization of 5′ non-coding sequences within the mammalian genomes. This methodology may be used for the identification of regulatory sequences in other lineages

Intracerebral delivery of Carboplatin in combination with either 6 MV Photons or monoenergetic synchrotron X-rays are equally efficacious for treatment of the F98 rat glioma.

International audienceABSTRACT: BACKGROUND: The purpose of the present study was to compare side-by-side the therapeutic efficacy of a 6-day infusion of carboplatin, followed by X-irradiation with either 6 MV photons or synchrotron X-rays, tuned above the K-edge of Pt, for treatment of F98 glioma bearing rats. METHODS: Carboplatin was administered intracerebrally (i.c.) to F98 glioma bearing rats over 6 days using AlzetTM osmotic pumps starting 7 days after tumor implantation. Radiotherapy was delivered in a single 15 Gy fraction on day 14 using a conventional 6 MV linear accelerator (LINAC) or 78.8 keV synchrotron X-rays. RESULTS: Untreated control animals had a median survival time (MeST) of 33 days. Animals that received either carboplatin alone or irradiation alone with either 78.8 keV or 6 MV had a MeSTs 38 and 33 days, respectively. Animals that received carboplatin in combination with X-irradiation had a MeST of > 180 days with a 55% cure rate, irrespective of whether they were irradiated with either 78.8 KeV synchrotron X-rays or 6MV photons. CONCLUSIONS: These studies have conclusively demonstrated the equivalency of i.c. delivery of carboplatin in combination with X-irradiation with either 6 MV photons or synchrotron X-rays

Deep UV resonant Raman spectroscopy for photodamage characterization in cells

We employed deep UV (DUV) Raman spectroscopy for characterization of molecular photodamage in cells. 244 nm light excitation Raman spectra were measured for HeLa cells exposed to the excitation light for different durations. In the spectra obtained with the shortest exposure duration (0.25 sec at 16 µW/µm2 irradiation), characteristic resonant Raman bands of adenine and guanine at 1483 cm−1 and tryptophan and tyrosine at 1618 cm−1 were clearly visible. With increasing exposure duration (up to 12.5 sec), these biomolecular Raman bands diminished, while a photoproduct Raman band at 1611 cm−1 grew. By exponential function fitting analyses, intensities of these characteristic three bands were correlated with sample exposure duration at different intensities of excitation light. We then suggest practical excitation conditions effective for DUV Raman observation of cells without photodamage-related spectral distortion

Solvent Exposure Associated with Single Abasic Sites Alters the Base Sequence Dependence of Oxidation of Guanine in DNA in GG Sequence Contexts

The effect of exposure of guanine in double-stranded oligonucleotides to aqueous solvent during oxidation by one-electron oxidants was investigated by introducing single synthetic tetrahydrofuran-type abasic sites (Ab) either adjacent to or opposite tandem GG sequences. The selective oxidation of guanine was initiated by photoexcitation of the aromatic sensitizers riboflavin and a pyrene derivative, and by the relatively small negatively charged carbonate radical anion. The relative rates of oxidation of the 5′- and 3′ side G in runs of 5′⋅⋅⋅GG⋅⋅⋅ (evaluated by standard hot alkali treatment of the damaged DNA strand followed by high resolution gel electrophoresis of the cleavage fragments) are markedly affected by adjacent abasic sites either on the same or opposite strand. For example, in fully double-stranded DNA or one with an Ab adjacent to the 5′-G, the 5′-G/3′-G damage ratio is ≥4, but is inverted (<1.0) with the Ab adjacent to the 3′-G. These striking effects of Ab are attributed to the preferential localization of the “hole” on the most solvent-exposed guanine regardless of the size, charge, or reduction potential of the oxidizing species