Project description:The data set contains MS/MS data on teeth extracts for Ancient DNA teeth samples ran in both positive and Negative ionization modes
Project description:Development of high resolution/accurate mass liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) methodology enables the characterization of covalently modified DNA induced by interaction with genotoxic agents in complex biological samples. Constant neutral loss monitoring of 2´-deoxyribose or the nucleobases using data-dependent acquisition represents a powerful approach for the unbiased detection of DNA modifications (adducts). The lack of available bioinformatics tools necessitates manual processing of acquired spectral data and hampers high throughput application of these techniques. To address this limitation, we present an automated workflow for the detection and curation of putative DNA adducts by using diagnostic fragmentation filtering of LC-MS/MS experiments within the open-source software MZmine. The workflow utilizes a new feature detection algorithm, DFBuilder, which employs diagnostic fragmentation filtering using a user-defined list of fragmentation patterns to reproducibly generate feature lists for precursor ions of interest. The DFBuilder feature detection approach readily fits into a complete small molecule discovery workflow and drastically reduces the processing time associated with analyzing DNA adductomics results. We validate our workflow using a mixture of authentic DNA adduct standards and demonstrate the effectiveness of our approach by reproducing and expanding the results of a previously published study of colibactin-induced DNA adducts. The reported workflow serves as a technique to assess the diagnostic potential of novel fragmentation pattern combinations for the unbiased detection of chemical classes of interest.
Project description:Crosslinking-MS analysis of sulfo-SDA crosslinked fission yeast condensin-DNA samples in the initial binding state (absence of nucleotide) and in the DNA gripping state (in the presence of ADP•BeF3)
Project description:Non-canonical DNA structures such as G-quadruplex (G4) and i-motif (iM) are formed at the guanine- and cytosine-rich sequences, respectively, and prohibit DNA replication and transcription. The formation and resolution of these non-canonical structures are therefore required to be dynamically regulated by either physiological conditions or factors able to bind the G4 and iM structures. Although many G4 binding proteins responsible for tuning of the G4 structure have been discovered, understanding of structural regulation of the iM structure by iM binding proteins is far less behind. Here, we developed a protein-labeling DNA probe bearing an alkyne moiety through a reactive tosylate linker for proximity labeling of nucleic acid-binding proteins and searched for iM binding proteins. The proteome analyses of the captured proteins suggested new candidates that potentially bind the iM structure, in addition to the known iM binders.
Project description:The prevailing views as to the form, function, and regulation of genomic methylation patterns have their origin many years in the past, at a time when the structure of the mammalian genome was only dimly perceived, when the number of protein-encoding mammalian genes was believed to be at least five times greater than the actual number, and when it was not understood that only ~10% of the genome is under selective pressure and likely to have biological function. We use more recent findings from genome biology and whole-genome methylation profiling to provide a reappraisal of the shape of genomic methylation patterns and the nature of the changes that they undergo during gametogenesis and early development. We observe that the sequences that undergo deep changes in methylation status during early development are largely sequences without regulatory function. We also discuss recent findings that begin to explain the remarkable fidelity of maintenance methylation. Rather than a general overview of DNA methylation in mammals (which has been the subject of many reviews), we present a new analysis of the distribution of methylated CpG dinucleotides across the multiple sequence compartments that make up the mammalian genome, and we offer an updated interpretation of the nature of the changes in methylation patterns that occur in germ cells and early embryos. We discuss the cues that might designate specific sequences for demethylation or de novo methylation during development, and we summarize recent findings on mechanisms that maintain methylation patterns in mammalian genomes. We also describe the several human disorders, each very different from the other, that are caused by mutations in DNA methyltransferase genes.
Project description:DNA polymerase nu (POLN or pol nu) is a newly discovered A family polymerase that generates a high error rate when incorporating nucleotides opposite dG; its translesion DNA synthesis (TLS) capability has only been demonstrated for high fidelity replication bypass of thymine glycol lesions. In the current investigation, we describe a novel TLS substrate specificity of pol nu, demonstrating that it is able to bypass exceptionally large DNA lesions whose linkages are through the DNA major groove. Specifically, pol nu catalyzed efficient and high fidelity TLS past peptides linked to N(6)-dA via a reduced Schiff base linkage with a gamma-hydroxypropano-dA. Additionally, pol nu could bypass DNA interstrand cross-links with linkage between N(6)-dAs in complementary DNA strands. However, the chemically identical DNA--peptide and DNA interstrand cross-links completely blocked pol nu when they were located in the minor groove via a N(2)-dG linkage. Furthermore, we showed that pol nu incorporated a nucleotide opposite the 1,N(6)-etheno-dA (epsilondA) in an error-free manner and (+)-trans-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide-dA [(+)-BPDE-dA] in an error-prone manner, albeit with a greatly reduced capability. Collectively, these data suggest that although pol nu bypass capacity cannot be generalized to all major groove DNA adducts, this polymerase could be involved in TLS when genomic replication is blocked by extremely large major groove DNA lesions. In view of the recent observation that pol nu may have a role in cellular tolerance to DNA cross-linking agents, our findings provide biochemical evidence for the potential functioning of this polymerase in the bypass of some DNA-protein and DNA-DNA cross-links.
Project description:BackgroundAlternative DNA conformations are of particular interest as potential signals to mark important sites on the genome. The structural variability of CA microsatellites is particularly pronounced; these are repetitive poly(CA). poly(TG) DNA sequences spread in all eukaryotic genomes as tracts of up to 60 base pairs long. Many in vitro studies have shown that the structure of poly(CA). poly(TG) can vary markedly from the classical right handed DNA double helix and adopt diverse alternative conformations. Here we have studied the mechanism of formation and the structure of an alternative DNA structure, named Form X, which was observed previously by polyacrylamide gel electrophoresis of DNA fragments containing a tract of the CA microsatellite poly(CA). poly(TG) but had not yet been characterized.ResultsFormation of Form X was found to occur upon reassociation of the strands of a DNA fragment containing a tract of poly(CA). poly(TG), in a process strongly stimulated by the nuclear proteins HMG1 and HMG2. By inserting Form X into DNA minicircles, we show that the DNA strands do not run fully side by side but instead form a DNA knot. When present in a closed DNA molecule, Form X becomes resistant to heating to 100 degrees C and to alkaline pH.ConclusionsOur data strongly support a model of Form X consisting in a DNA loop at the base of which the two DNA duplexes cross, with one of the strands of one duplex passing between the strands of the other duplex, and reciprocally, to form a semicatenated DNA junction also called a DNA hemicatenane.
Project description:Nucleic acids, due to their structural and chemical properties, can form double-stranded secondary structures that assist the transfer of genetic information and can modulate gene expression. However, the nucleotide sequence alone is insufficient in explaining phenomena like intron-exon recognition during RNA processing. This raises the question whether nucleic acids are endowed with other attributes that can contribute to their biological functions. In this work, we present a calculation of thermodynamic stability of DNA/DNA and mRNA/DNA duplexes across the genomes of four species in the genus Saccharomyces by nearest-neighbor method. The results show that coding regions are more thermodynamically stable than introns, 3'-untranslated regions and intergenic sequences. Furthermore, open reading frames have more stable sense mRNA/DNA duplexes than the potential antisense duplexes, a property that can aid gene discovery. The lower stability of the DNA/DNA and mRNA/DNA duplexes of 3'-untranslated regions and the higher stability of genes correlates with increased mRNA level. These results suggest that the thermodynamic stability of DNA/DNA and mRNA/DNA duplexes affects mRNA transcription.