Repair of UV-induced DNA damage requires chromatin remodeling. How repair is initiated in chromatin remains largely unknown. We recently demonstrated that global genome-nucleotide excision repair (GG-NER) in chromatin is organized into domains in relation to open reading frames. Here, we define these domains, identifying the genomic locations from which repair is initiated. By examining DNA damage-induced changes in the linear structure of nucleosomes at these sites, we demonstrate how chromatin remodeling is initiated during GG-NER. In undamaged cells, we show that the GG-NER complex occupies chromatin, establishing the nucleosome structure at these genomic locations, which we refer to as GG-NER complex binding sites (GCBSs). We demonstrate that these sites are frequently located at genomic boundaries that delineate chromosomally interacting domains (CIDs). These boundaries define domains of higher-order nucleosome-nucleosome interaction. We demonstrate that initiation of GG-NER in chromatin is accompanied by the disruption of dynamic nucleosomes that flank GCBSs by the GG-NER complex.

The rates at which lesions are removed by DNA repair can vary widely throughout the genome with important implications for genomic stability. To study this, we measured the distribution of nucleotide excision repair (NER) rates for UV-induced lesions throughout the budding yeast genome. By plotting these repair rates in relation to genes and their associated flanking sequences, we reveal that in normal cells, genomic repair rates display a distinctive pattern, suggesting that DNA repair is highly organised within the genome. Furthermore, by comparing genome-wide DNA repair rates in wild-type cells, and cells defective in the global genome-NER (GG-NER) sub-pathway, we establish how this alters the distribution of NER rates throughout the genome. We also examined the genomic locations of GG-NER factor binding to chromatin before and after UV irradiation revealing that GG-NER is organised and initiated from specific genomic locations. At these sites, chromatin occupancy of the histone acetyl transferase Gcn5 is controlled by the GG-NER complex, which regulates histone H3 acetylation and chromatin structure, thereby promoting efficient DNA repair of UV-induced lesions. Chromatin remodeling during the GG-NER process is therefore organized into these genomic domains. Importantly, loss of Gcn5, significantly alters the genomic distribution of NER rates, a finding that has important implications for the effects of chromatin modifiers on the distribution of mutations that arise throughout the genome.

Genotoxins cause DNA damage, which can result in genomic instability. The genetic changes induced have far-reaching consequences, often leading to diseases such as cancer. A wide range of genotoxins exists, including radiations and chemicals found naturally in the environment and in man-made forms created by human activity across a variety of industries. Genomic technologies offer the possibility of unravelling the mechanisms of genotoxicity, including the repair of genetic damage, enhancing our ability to develop, test and safely use existing and novel materials. We have developed 3D-DIP-Chip, a microarray-based method to measure the prevalence of genomic genotoxin-induced DNA damage. We demonstrate the measurement of both physical and chemical induced DNA damage spectra, integrating the analysis of these with the associated changes in histone acetylation induced in the epigenome. We discuss the application of the method in the context of basic and translational sciences.

ChIP-chip is a microarray based technology for determining the genomic locations of chromatin bound factors of interest, such as proteins. Standard ChIP-chip analyses employ peak detection methodologies to generate lists of genomic binding sites. No previously published method exists to enable comparative analyses of enrichment levels derived from datasets examining different experimental conditions. This restricts the use of the technology to binary comparisons of presence or absence of features between datasets. Here we present the R package Sandcastle — S oftware for the A nalysis and N ormalisation of D ata from C hIP-chip A ssay S of T wo or more L inked E xperiments — which allows for comparative analyses of data from multiple experiments by normalising all datasets to a common background. Relative changes in binding levels between experimental datasets can thus be determined, enabling the extraction of latent information from ChIP-chip experiments. Novel enrichment detection and peak calling algorithms are also presented, with a range of graphical tools, which facilitate these analyses. The software and documentation are available for download from http://reedlab.cardiff.ac.uk/sandcastle.

BackgroundMany chemotherapy agents act by inducing DNA damage, leading to cell death. Resistance and treatment failure limits the efficacy of these agents, and repair of the DNA damage is the commonest cause of resistance. By combining DNA immunoprecipitation with DNA microarray technology, we have developed a novel method to detect, quantify, and localise DNA damage at high resolution throughout the human genome.MethodsWe developed this assay initially in the model yeast organism Saccharomyces cerevisiae, before further development in human dermal fibroblast cells. Cellular DNA was treated in vivo or naked DNA treated in vitro with cisplatin, oxaliplatin, or ultraviolet (UV) irradiation. Using antibodies against cisplatin-modified DNA or cyclobutane pyrimidine dimers, we captured damaged DNA fragments through DNA immunoprecipitation. DNA samples were then amplified, labelled and hybridised to custom-designed DNA microarrays to generate high-resolution genome-wide profiles for DNA damage.FindingsGenome-wide cisplatin, oxaliplatin, and UV-induced damage profiles were generated in human cells and were consistent, reproducible, and correlated well with mathematical models predicting sites of damage induction developed in our laboratory (r=0·7–0·8). Using standard chromatin immunoprecipitation-DNA microarray experiments, we generated genome-wide epigenetic profiles showing increased H3K14 acetylation after platinum exposure. By applying novel bioinformatic outlier detection methods, we identified genomic regions where differences occured between in-vitro and in-vivo damage profiles, and we demonstrated how this information could be integrated and correlated with epigenetic changes.InterpretationWe validated a novel technique using DNA microarrays to sensitively measure DNA damage and repair at high resolution throughout the human genome, and we used the platinum analogue chemotherapy agents and UV irradiation as a model for damage induction. This technology can be applied to other DNA damaging agents and to clinical samples taken from patients after treatment. We believe that by applying this assay, and using our bioinformatic approaches for integrating genomic and epigenetic datasets, we will be able to detect functional biomarkers and genetic signatures predictive of response or toxicity, allowing us to optimise and stratify the use of current therapies. We are applying these assays to clinical samples.FundingUK Medical Research Council, Velindre NHS Trust Charitable Funds.

A technique has emerged over the past decade combining chromatin immunoprecipitation with DNA microarray analysis. This is a powerful and sensitive strategy that has been used extensively to characterise protein interactions with chromatin and epigenetic changes such as acetylation and methylation throughout the genome of different organisms. This technique has revolutionised our understanding of molecular genomics, continues to be widely used and is currently being applied in novel areas of cancer research. In this publication we review the historical context of this technology and offer current and future perspectives on how this technique is currently being developed and modified to allow its use in novel areas of research. We discuss the potential for this technique and its ongoing important role in biological research particularly in relation to cancer research. We also offer insight into the potential clinical application of this technology in stratified medicine, particularly in the field of cancer therapy.

Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER) in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes.

DNA is not chemically inert but faces constant challenges to its stability. One of these is the fusion of adjacent pyrimidine bases by ultra violet (UV) radiation to create cyclobutane pyrimidine dimers (CPDs). Numerous methods of DNA repair have evolved within cells, of which nucleotide excision repair (NER) is responsible for the removal of CPDs and other bulky adducts. To investigate this and other repair pathways various techniques have been developed to detect DNA damage at low resolutions in whole genomes or high resolutions over small sections of a genome. We have developed a novel microarray based method for the genome wide high resolution analysis of DNA damage in yeast which combines the advantages of these, allowing detailed measurement of repair across entire genomes. A program has been written to predict the expected CPD formation based on sequence; this has shown that the genome wide damage detection method is accurate. Additionally, ChIPchip has been used to determine the binding positions of proteins involved in NER and analyse histone modifications after damage induction. Combining these datasets allows protein binding and acetylation levels to be correlated with repair rates. These datasets require bioinformatic tools to analyse and extract results. I have developed a suite of novel tools to process, normalise, display and interrogate these datasets including a new normalisation method which allows accurate comparisons to be made between different factors, revealing changes in acetylation profiles following UV and between different mutant strains, a peak detection method to distinguish protein binding peaks from a background of nonbound regions, revealing many novel binding sites for proteins such as Abf1 and Rad16, and graphical displays to determine patterns that occur at multiple positions throughout genomes, revealing patterns of varying repair rates at regions such as centromeres and telomeres.

Knowing where DNA damage occurs within a genome is crucial to the understanding of the potential genetic consequences of such damage. We here reported a new development based on microarray technology which uses ultraviolet light induced DNA damage as a paradigm to determine the position and frequency of DNA damage and its subsequent repair throughout the entire yeast genome (1). Analyses of genome-wide DNA repair can be undertaken, alongside examination of the DNA damage-induced changes in chromatin that facilitate repair. The approach should be applicable to examining a spectrum of DNA damages provided either the antibodies or the tagged DNA damage recognition enzymes are available to immunoprecipitate those specific DNA damages, and provided that any of those damages that block PCR can be repaired to enable PCR. The method is applicable to studies with human cells via the available higher resolution human microarrays and experiments are underway to optimize this approach with such cells. DNA samples prepared for this microarray-based approach can be readily processed for high throughput sequencing analysis if this detail is required, or if microarrays are unavailable for the organism of interest. The technique has substantial implications for monitoring the DNA targets during genotoxicity testing. It will enable an estimation of the target sequences for DNA damage and whether these interactions persist in cells; an important facet if one considers threshold levels for DNA damage along the lines employed in estimating the risk associated with radiation exposure. A patent application covering the approach has entered the international PCT phase.

DNA damage occurs via endogenous and exogenous genotoxic agents and compromises a genome's integrity. Knowing where damage occurs within a genome is crucial to understanding the repair mechanisms which protect this integrity. This paper describes a new development based on microarray technology which uses ultraviolet light induced DNA damage as a paradigm to determine the position and frequency of DNA damage and its subsequent repair throughout the entire yeast genome.