A novel method for the genome-wide high resolution analysis of DNA damage

Teng, Yumin; Bennett, Mark; Evans, Katie; Zhuang-Jackson, Huayun; Higgs, Andy; Reed, Simon H.; Waters, Raymond

doi:10.1093/nar/gkq1036

Cited by 41 publications

(46 citation statements)

References 13 publications

(15 reference statements)

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“…Unlike uracil incorporation, CPD and 6-4pp dimers were uniformly distributed across the genome, consistent with previous studies showing uniform densities of CPD formation (Teng et al 2011). Future application of Excision-seq to map UV photoproducts may uncover more subtle patterns associated with genomic features, including nucleosome positions or other structural features.…”

Section: Discussionsupporting

confidence: 87%

“…If a similar mechanism for uracil content variation operates in human cells, it might inherently counteract common chemotherapies such as 5-fluorouracil, which raise dUTP levels to promote cycles of incorporation and repair, causing DNA damage and apoptosis. Elucidation of the mechanism underlying uracil content variation may therefore have implications for the design and delivery of therapeutics that cause nucleotide pool imbalances to promote cell death.Unlike uracil incorporation, CPD and 6-4pp dimers were uniformly distributed across the genome, consistent with previous studies showing uniform densities of CPD formation (Teng et al 2011). Future application of Excision-seq to map UV photoproducts may uncover more subtle patterns associated with genomic features, including nucleosome positions or other structural features.…”

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confidence: 87%

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High resolution mapping of modified DNA nucleobases using excision repair enzymes

et al. 2014

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The incorporation and creation of modified nucleobases in DNA have profound effects on genome function. We describe methods for mapping positions and local content of modified DNA nucleobases in genomic DNA. We combined in vitro nucleobase excision with massively parallel DNA sequencing (Excision-seq) to determine the locations of modified nucleobases in genomic DNA. We applied the Excision-seq method to map uracil in E. coli and budding yeast and discovered significant variation in uracil content, wherein uracil is excluded from the earliest and latest replicating regions of the genome, possibly driven by changes in nucleotide pool composition. We also used Excision-seq to identify sites of pyrimidine dimer formation induced by UV light exposure, where the method could distinguish between sites of cyclobutane and 6-4 photoproduct formation. These UV mapping data enabled analysis of local sequence bias around pyrimidine dimers and suggested a preference for an adenosine downstream from 6-4 photoproducts. The Excision-seq method is broadly applicable for high precision, genome-wide mapping of modified nucleobases with cognate repair enzymes.[Supplemental material is available for this article.]Many different modifications of the four primary DNA nucleobases expand the chemical diversity of DNA and have profound effects on genome function. Intrinsic modifications (e.g., 5-methylcytosine and uracil) are integral to genetic and epigenetic regulation. Extrinsic modifications (e.g., pyrimidine dimers and nucleobase oxidation) arise from environmental exposures and can initiate aberrant cell growth or death. A detailed understanding of intrinsic and extrinsic nucleobase modification is necessary for a complete view of genetic and epigenetic regulation, but a global picture of how nucleobase modifications are created, maintained, and repaired, and how their spatial distribution impacts genome function, is lacking.Incorporation of uracil into DNA creates detrimental or beneficial mutations, depending on context. To sustain DNA replication, cells must synthesize or scavenge precursors to accumulate a pool of nucleotide triphosphates. A key step of thymidine triphosphate (TTP) synthesis is catalyzed by thymidylate synthase, which converts dUMP to dTMP using tetrahydrofolate as a methyl donor. One branch of the TTP biosynthetic pathway uses dUTP as an intermediate, which can be incorporated into DNA in the form of A:U base pairs. The upstream production of dUMP is catalyzed by deamination of dCMP by deoxycytidylate deaminase or pyrophosphorolysis of dUTP by the dUTP pyrophosphatase (Dut1). Dut1 is essential for viability and normal nucleotide metabolism: In the absence of Dut1, cells simultaneously accumulate dUTP and deplete TTP pools (Gadsden et al. 1993), causing a futile cycle of uracil incorporation and repair that leads to extensive DNA damage (Kavli et al. 2007).Uracil in DNA is removed by uracil DNA glycosylase (UDG) enzymes, which scan double-stranded DNA for uracil and cleave its glycosidic bond (Krokan et al. 200...

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Section: Discussionsupporting

confidence: 87%

supporting

confidence: 87%

High resolution mapping of modified DNA nucleobases using excision repair enzymes

et al. 2014

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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.

128

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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“…Thus, any method used to map DNA repair must yield a high signal to noise ratio and high sensitivity. Although genome-wide maps of UV damage distribution (Teng et al 2011;Bryan et al 2014;Zavala et al 2014;Powell et al 2015) and single-gene analyses of excision repair in human cells and yeast are available (Pfeifer et al 1991;Tornaletti and Pfeifer 1994;Denissenko et al 1996;Li et al 2000Li et al , 2014, methods for genome-wide detection of excision repair at nucleotide resolution have not been reported.…”

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confidence: 99%

Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution

et al. 2015

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We developed a method for genome-wide mapping of DNA excision repair named XR-seq (excision repair sequencing). Human nucleotide excision repair generates two incisions surrounding the site of damage, creating an ∼30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of repair of two UV-induced DNA damages in human cells: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs]. In wild-type cells, CPD repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled excision repair or general excision repair isolated the contribution of each pathway to the overall repair pattern and showed that transcription-coupled repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled repair at sites of divergent gene promoters and bidirectional enhancer RNA (eRNA) production at enhancers. XR-seq data also uncovered the repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA repair in human cells.

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A novel method for the genome-wide high resolution analysis of DNA damage

Cited by 41 publications

References 13 publications

High resolution mapping of modified DNA nucleobases using excision repair enzymes

High resolution mapping of modified DNA nucleobases using excision repair enzymes

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

Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution

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