3D-DIP-Chip: a microarray-based method to measure genomic DNA damage

Powell, J.R.; Bennett, Mark; Evans, Katie; Yu, Shirong; Webster, Richard; Waters, Raymond; Skinner, Nigel; Reed, Simon H.

doi:10.1038/srep07975

Cited by 34 publications

(41 citation statements)

References 18 publications

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“…CPD formation is dictated mostly by sequence, and previous reports of the genome-wide distributions of this type of damage showed a relatively widespread signal that does not follow the enrichment patterns we observed (27,28). Furthermore, if higher initial repair were solely explained by higher levels of damage, we would expect similar enrichments in all time points.…”

Section: Regions Repaired Later Are Associated With Higher Mutagenesismentioning

confidence: 51%

Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis

Adar

Lieb

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

156

226

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We recently developed a high-resolution genome-wide assay for mapping DNA excision repair named eXcision Repair-sequencing (XR-seq) and have now used XR-seq to determine which regions of the genome are subject to repair very soon after UV exposure and which regions are repaired later. Over a time course, we measured repair of the UV-induced damage of cyclobutane pyrimidine dimers (CPDs) (at 1, 4, 8, 16, 24, and 48 h) and (6-4)pyrimidinepyrimidone photoproducts [(6-4)PPs] (at 5 and 20 min and 1, 2, and 4 h) in normal human skin fibroblasts. Each type of damage has distinct repair kinetics. The (6-4)PPs are detected as early as 5 min after UV treatment, with the bulk of repair completed by 4 h. Repair of CPDs, which we previously showed is intimately coupled to transcription, is slower and in certain regions persists even 2 d after UV irradiation. We compared our results to the Encyclopedia of DNA Elements data regarding histone modifications, chromatin state, and transcription. For both damage types, and for both transcription-coupled and general excision repair, the earliest repair occurred preferentially in active and open chromatin states. Conversely, repair in regions classified as "heterochromatic" and "repressed" was relatively low at early time points, with repair persisting into the late time points. Damage that remains during DNA replication increases the risk for mutagenesis. Indeed, laterepaired regions are associated with a higher level of cancer-linked mutations. In summary, we show that XR-seq is a powerful approach for studying relationships among chromatin state, DNA repair, genome stability, mutagenesis, and carcinogenesis.DNA repair | DNA damage | chromatin | transcription | mutation D NA damage blocks transcription and replication and compromises the integrity of the genome. Bulky adducts in DNA, the focus of this work, are caused by a variety of genotoxic agents including UV radiation in sunlight. In human cells, this damage is removed exclusively by the nucleotide excision repair mechanism (1-3). In nucleotide excision repair, a single-stranded oligomer that contains the bulky adduct is excised by dual incisions bracketing the lesion. The resulting gap is filled in by DNA polymerases, and the newly synthesized repair patch is then sealed by DNA ligase. In general excision repair, damage recognition is achieved by the repair factors xeroderma pigmentosum (XP) complementation group C (XPC) together with replication protein A (RPA) and XPA (4-6). In DNA that is being actively transcribed, damage recognition can also be achieved by a stalled RNA polymerase II, which, with the aid of the Cockayne Syndrome B (CSB) protein, accelerates the recruitment of the excision repair factors. This recognition process leads to transcription-coupled repair (7,8). The subsequent dual-incision reaction is carried out by the core excision repair factors RPA, XPA, transcription factor II H (TFIIH), XPG, and XPF-ERCC1, releasing an excised oligomer that is 24-32 nucleotides (nt) in length (6, 9).The mechanism of DNA...

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Section: Regions Repaired Later Are Associated With Higher Mutagenesismentioning

confidence: 51%

Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis

Adar

Lieb

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

156

226

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“…S8). Such a nucleosome photo-footprint was identified in mammalian chromatin nearly 30 y ago (11), but had been missed by previous genome-wide studies of CPD-formation (15,16,18,25). Nucleosomal DNA with an inward rotational setting is likely protected from UV damage as a result of constraints on DNA bending and flexibility imposed by the nucleosome structure (22).…”

Section: Discussionmentioning

confidence: 99%

“…8); however, past biochemical and cellular studies have suggested that nucleosomes and DNA-binding proteins may significantly alter the formation of UV damage (11)(12)(13)(14). Previous attempts to measure initial CPD levels using microarray-based methods (15)(16)(17) suggested that UV damage formation is primarily influenced by the DNA sequence. For example, CPDs occur most frequently at TT dipyrimidines, followed by TC, CT, and CC.…”

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confidence: 99%

Chromosomal landscape of UV damage formation and repair at single-nucleotide resolution

Mao

Smerdon

Roberts

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

134

236

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UV-induced DNA lesions are important contributors to mutagenesis and cancer, but it is not fully understood how the chromosomal landscape influences UV lesion formation and repair. Genome-wide profiling of repair activity in UV irradiated cells has revealed significant variations in repair kinetics across the genome, not only among large chromatin domains, but also at individual transcription factor binding sites. Here we report that there is also a striking but predictable variation in initial UV damage levels across a eukaryotic genome. We used a new high-throughput sequencing method, known as CPD-seq, to precisely map UV-induced cyclobutane pyrimidine dimers (CPDs) at single-nucleotide resolution throughout the yeast genome. This analysis revealed that individual nucleosomes significantly alter CPD formation, protecting nucleosomal DNA with an inward rotational setting, even though such DNA is, on average, more intrinsically prone to form CPD lesions. CPD formation is also inhibited by DNAbound transcription factors, in effect shielding important DNA elements from UV damage. Analysis of CPD repair revealed that initial differences in CPD damage formation often persist, even at later repair time points. Furthermore, our high-resolution data demonstrate, to our knowledge for the first time, that CPD repair is significantly less efficient at translational positions near the dyad of strongly positioned nucleosomes in the yeast genome. These findings define the global roles of nucleosomes and transcription factors in both UV damage formation and repair, and have important implications for our understanding of UV-induced mutagenesis in human cancers.DNA repair | DNA damage | nucleosome | chromatin | transcription factor U ltraviolet (UV) light causes extensive damage to DNA by inducing the formation of cyclobutane pyrimidine dimers (CPDs) and, to a lesser extent, 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs). If unrepaired, these DNA lesions block normal DNA replication and are major contributors to mutagenesis in skin cancers (1). CPDs and 6-4PPs are primarily repaired in cells by the nucleotide excision repair (NER) pathway (1). CPD lesions in actively transcribed strands (TS) of DNA are repaired rapidly by the transcription coupled-NER (TC-NER) branch of this repair pathway, which is triggered by RNA polymerase II stalling at UV damage (2, 3). In contrast, CPD lesions in the remainder of the genome are repaired by the global genome NER (GG-NER) subpathway. Differences in repair rates between transcribed and nontranscribed DNA, and between accessible and inaccessible chromatin domains, have been invoked to explain the mutational heterogeneity in many cancer genomes (4-6). To gain new insight into the mutational processes that lead to human cancer, however, a more detailed understanding is needed of the complex interplay of UV damage formation and repair across the genome.Our understanding of how NER operates in different sequence and chromatin contexts has been aided by two recent genome-wide surveys of NER ac...

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“…To tackle this issue, we developed a genome-wide DNA repair assay based on ChIP-chip, referred to as 3D-DIP-Chip Powell et al 2015). The method permits the calculation of the relative repair rates at individual sites throughout the genome.…”

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confidence: 99%

Global genome nucleotide excision repair is organized into domains that promote efficient DNA repair in chromatin

Evans

Eijk

et al. 2016

Genome Res.

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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 organized 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) subpathway, 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 organized 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; this has implications for the effects of chromatin modifiers on the distribution of mutations that arise throughout the genome.

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3D-DIP-Chip: a microarray-based method to measure genomic DNA damage

Cited by 34 publications

References 18 publications

Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis

Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis

Chromosomal landscape of UV damage formation and repair at single-nucleotide resolution

Global genome nucleotide excision repair is organized into domains that promote efficient DNA repair in chromatin

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