Simultaneous measurement of DNA repair activities and mRNA abundance in single cells
Ontology highlight
ABSTRACT:
ORGANISM(S): synthetic construct Homo sapiens
PROVIDER: GSE130117 | GEO | 2019/10/02
REPOSITORIES: GEO
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Project description:Simultaneous measurement of DNA repair activities and mRNA abundance in single cells
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Project description:The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates are either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exists to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase, hyTDG, which is comprised of a 29-amino acid sequence from human thymine DNA glycosylase linked to a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hyTDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC-MS/MS identification and quantification. Using this approach, we have measured for the first time the levels of total Uracil (U), U:G and T:G in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.
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Project description:Here we present a new version of the DamID method that allows efficient capturing of protein-DNA interactions and mRNA output from the same single cell. With this protocol, we have measured the direct impact of spatial genome positioning and chromatin accessibility on mRNA output in human haploid cells
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Project description:Shimoni2009 - Escherichia Coli SOS
Simple model, involving only the basic components of the circuit, sufficient to explain the peaks in the promoter activities of recA and lexA.
This model is described in the article:
Stochastic analysis of the SOS response in Escherichia coli.
Shimoni Y, Altuvia S, Margalit H, Biham O
PloS one. 2009; 4(5):e5363
Abstract:
BACKGROUND: DNA damage in Escherichia coli evokes a response mechanism called the SOS response. The genetic circuit of this mechanism includes the genes recA and lexA, which regulate each other via a mixed feedback loop involving transcriptional regulation and protein-protein interaction. Under normal conditions, recA is transcriptionally repressed by LexA, which also functions as an auto-repressor. In presence of DNA damage, RecA proteins recognize stalled replication forks and participate in the DNA repair process. Under these conditions, RecA marks LexA for fast degradation. Generally, such mixed feedback loops are known to exhibit either bi-stability or a single steady state. However, when the dynamics of the SOS system following DNA damage was recently studied in single cells, ordered peaks were observed in the promoter activity of both genes (Friedman et al., 2005, PLoS Biol. 3(7):e238). This surprising phenomenon was masked in previous studies of cell populations. Previous attempts to explain these results harnessed additional genes to the system and deployed complex deterministic mathematical models that were only partially successful in explaining the results.
PRINCIPAL FINDINGS: Here we apply stochastic methods, which are better suited for dynamic simulations of single cells. We show that a simple model, involving only the basic components of the circuit, is sufficient to explain the peaks in the promoter activities of recA and lexA. Notably, deterministic simulations of the same model do not produce peaks in the promoter activities.
SIGNIFICANCE: We conclude that the double negative mixed feedback loop with auto-repression accounts for the experimentally observed peaks in the promoter activities. In addition to explaining the experimental results, this result shows that including additional regulations in a mixed feedback loop may dramatically change the dynamic functionality of this regulatory module. Furthermore, our results suggests that stochastic fluctuations strongly affect the qualitative behavior of important regulatory modules even under biologically relevant conditions, thus emphasizing the importance of stochastic analysis of regulatory circuits.
This model is hosted on BioModels Database and identified by: MODEL2937159804.
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2005-01-01 | MODEL2937159804 | BioModels
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2022-05-12 | GSE184372 | GEO
Project description:DNA methylation plays important roles in foreign DNA defense, mismatch repair, and gene regulation in prokaryotic genomes. Existing methods for DNA methylation detection using next-generation sequencing (NGS) are incapable of simultaneously detecting multiple types of DNA methylation. Here, we present nitrite treatment followed by sequencing (NT-seq), a sequencing method to simultaneously detect adenine and cytosine methylation. We demonstrated that NT-seq reliably detects three types of methylation motifs in E. coli and H. pylori genomes. We further applied NT-seq to a microbial community standard for de novo methylation motif discovery. Finally, by coupling methyl DNA immunoprecipitation and NT-seq (DIP-NT-seq), we showed that 6mA could be accurately mapped at single-base resolution in the bacterial and eukaryotic genomes. NT-seq thus provides a simple and reliable solution for detecting multiple types of DNA methylations.
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