No abstract

Environmental exposures, reactive by-products of cellular metabolism, and spontaneous deamination events result in a spectrum of DNA adducts that if un-repaired threaten genomic integrity by inducing mutations, increasing instability, and contributing to the initiation and progression of cancer. Assessment of DNA adducts in cells and tissues is critical for genotoxic and carcinogenic evaluation of chemical exposure and may provide insight into the etiology of cancer. Numerous methods to characterize the formation of DNA adducts and their retention for risk assessment have been developed. However, there are still significant drawbacks to the implementation and wide-spread use of these methods, because they often require a substantial amount of biological sample, highly specialized expertise and equipment, and depending on technique, may be limited to the detection and quantification of only a handful of DNA adducts at a time. There is a pressing need for high throughput, easy to implement assays that can assess a broad spectrum of DNA lesions, allowing for faster evaluation of chemical exposures and assessment of the retention of adducts in biological samples. Here, we describe a new methodology, Repair Assisted Damage Detection (RADD), which utilizes a DNA damage processing repair enzyme cocktail to detect and modify sites of DNA damage for a subsequent gap filling reaction that labels the DNA damage sites. This ability to detect and label a broad spectrum of DNA lesions within cells, offers a novel and easy to use tool for assessing levels of DNA damage in cells that have been exposed to environmental agents or have natural variations in DNA repair capacity.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

BackgroundActivation Induced cytidine Deaminase (AID) targets the immunoglobulin genes of activated B cells, where it converts cytidine to uracil to induce mutagenesis and recombination. While essential for immunoglobulin gene diversification, AID misregulation can result in genomic instability and oncogenic transformation. This is classically illustrated in Burkitt's lymphoma, which is characterized by AID-induced mutation and reciprocal translocation of the c-MYC oncogene with the IgH loci. Originally thought to be B cell-specific, AID now appears to be misexpressed in several epithelial cancers, raising the specter that AID may also participate in non-B cell carcinogenesis.MethodsThe mutagenic potential of AID argues for the existence of cellular regulators capable of repressing inappropriate AID expression. MicroRNAs (miRs) have this capacity, and we have examined the publically available human AID EST dataset for miR complementarities to the human AID 3'UTR. In this work, we have evaluated the capacity of two candidate miRs to repress human AID expression in MCF-7 breast carcinoma cells.ResultsWe have discovered moderate miR-155 and pronounced miR-93 complementary target sites encoded within the human AID mRNA. Luciferase reporter assays indicate that both miR-93 and miR-155 can interact with the 3'UTR of AID to block expression. In addition, over-expression of either miR in MCF-7 cells reduces endogenous AID protein, but not mRNA, levels. Similarly indicative of AID translational regulation, depletion of either miR in MCF-7 cells increases AID protein levels without concurrent increases in AID mRNA.ConclusionsTogether, our findings demonstrate that miR-93 and miR-155 constitutively suppress AID translation in MCF-7 cells, suggesting widespread roles for these miRs in preventing genome cytidine deaminations, mutagenesis, and oncogenic transformation. In addition, our characterization of an obscured miR-93 target site located within the AID 3'UTR supports the recent suggestion that many miR regulations have been overlooked due to the prevalence of truncated 3'UTR annotations.

Noncoding RNA (ncRNA) modulation of gene expression has now been ubiquitously observed across all domains of life. An increasingly apparent role of ncRNAs is to coordinate changes in gene expressions in response to environmental stress. Salmonella enterica, a common food-born pathogen, is known for its striking ability to survive, adapt, and thrive in various unfavourable environments which makes it a particularly difficult pathogen to eliminate as well as an interesting model in which to study ncRNA contributions to cellular stress response. Mounting evidence now suggests that small RNAs (sRNAs) represent key regulators of Salmonella stress adaptation. Approximately 50-500 nucleotides in length, sRNAs regulate gene expression through complementary base pairing with molecular targets and have recently been suggested to outnumber protein-coding genes in bacteria. In this work, we employ small RNA transcriptome sequencing to characterize changes in the sRNA profiles of Salmonella in response to desiccation. In all, we identify 102 previously annotated sRNAs significantly differentially expressed during desiccation; and excitingly, 71 novel sRNAs likewise differentially expressed. Small transcript northern blotting and qRT-PCRs confirm the identities and expressions of several of our novel sRNAs, and computational analyses indicate the majority are highly conserved and structurally related to characterized sRNAs. Predicted sRNA targets include several proteins necessary for desiccation survival and this, in part, suggests a role for desiccation-regulated sRNAs in this stress response. Furthermore, we find individual knockouts of two of the novel sRNAs identified herein, either sRNA1320429 or sRNA3981754, significantly impairs the ability of Salmonella to survive desiccation, confirming their involvements (and suggesting the potential involvements of other sRNAs we identify in this work) in the Salmonella response to desiccation.

BackgroundSomatic hypermutation introduces base substitutions into the rearranged and expressed immunoglobulin (Ig) variable regions to promote immunity. This pathway requires and is initiated by the Activation Induced Deaminase (AID) protein, which deaminates cytidine to produce uracils and UG mismatches at the Ig genes. Subsequent processing of uracil by mismatch repair and base excision repair factors contributes to mutagenesis. While selective for certain genomic targets, the chromatin modifications which distinguish hypermutating from non-hypermutating loci are not defined.Methodology/Principal FindingsHere, we show that AID-targeted loci in mammalian B cells contain ubiquitinated chromatin. Chromatin immunoprecipitation (ChIP) analysis of a constitutively hypermutating Burkitt's B cell line, Ramos, revealed the presence of monoubiquitinated forms of both histone H2A and H2B at two AID-associated loci, but not at control loci which are expressed but not hypermutated. Similar analysis using LPS activated primary murine splenocytes showed enrichment of the expressed VH and Sγ3 switch regions upon ChIP with antibody specific to AID and to monoubiquitinated H2A and H2B. In the mechanism of mammalian hypermutation, AID may interact with ubiquitinated chromatin because confocal immunofluorescence microscopy visualized AID colocalized with monoubiquitinated H2B within discrete nuclear foci.Conclusions/SignificanceOur results indicate that monoubiquitinated histones accompany active somatic hypermutation, revealing part of the histone code marking AID-targeted loci. This expands the current view of the chromatin state during hypermutation by identifying a specific nucleosome architecture associated with somatic hypermutation.

Genome sequences that contain tandem repeats of guanine can form stable four-stranded structures known as G-quadruplex, or G4 DNA. While the molecular mechanisms are not fully defined, such guanine-rich loci are prone to mutagenesis and recombination. Various repair pathways function to reduce the potential for genome instability by correcting base damage and replication errors; however, it is not yet fully defined how well these processes function at G4 DNA. One frequent form of base damage occurs from cytidine deamination, resulting in deoxyuracil and UG mismatches. In duplex and single-stranded DNA, uracil bases are recognised and excised by uracil glycosylases. Here, we tested the efficiency of uracil glycosylase activity in vitro on uracil bases located directly adjacent to guanine repeats and G4 DNA. We show that uracil excision by bacterial UDG and human hUNG2 is reduced at uracils positioned directly 5' or 3' of a guanine tetrad. Control reactions using oligonucleotides disrupted for G4 formation or reaction conditions that do not favour G4 formation resulted in full uracil excision activity. Based on these in vitro results, we suggest that folding of guanine-rich DNA into G4 DNA results in a DNA conformation that is resistant to uracil glycosylase-initiated repair and this has the potential to increase the risk of instability at guanine repeats in the genome.

Detection and characterization of DNA damage is essential for evaluating genotoxicity, monitoring DNA repair, developing biomarkers for exposures, and evaluating the efficacy of chemotherapies. These diverse applications for DNA damage measurements have spurred the continual development and refinement of methodologies for detecting, characterizing, and quantifying DNA damage from isolated DNA and in cells and tissues. Current damage detection methods cover a wide range of techniques from radiolabeling to mass spectrometry, and use of these techniques varies widely based on expense, expertise, and knowledge of adduct formation. More generalizable, easy-to-use methods for detecting and quantifying DNA damage are needed, and there has been an emergence of fluorescence-based methodologies to address this need. Developments in these fluorescence-based strategies are reviewed here.