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

Adar, Sheera; Hu, Jinchuan; Lieb, Jason D.; Sancar, Aziz

doi:10.1073/pnas.1603388113

Cited by 160 publications

(254 citation statements)

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“…Thus, by the sheer number of events, a process that itself is hardly mutagenic might become a significant cause for genome alterations. Interestingly, a recent study performed with mammalian cells indicates that, most likely due to delayed damage recognition, NER is slow in heterochromatic regions and leads to the accumulation of a significant number of mutations (35). In this sense, paired SSBs, whether they occur at high frequency in the same strand or at low frequency in opposite strands in close proximity, might be a major threat to genome stability.…”

Section: The Role Of Paired Ssbs On Opposite Strands In the Formation Ofmentioning

confidence: 99%

Repair of adjacent single-strand breaks is often accompanied by the formation of tandem sequence duplications in plant genomes

Schiml

Fauser

Puchta

2016

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

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Duplication of existing sequences is a major mechanism of genome evolution. It has been previously shown that duplications can occur by replication slippage, unequal sister chromatid exchange, homologous recombination, and aberrant double-strand break-induced synthesis-dependent strand annealing reactions. In a recent study, the abundant presence of short direct repeats was documented by comparative bioinformatics analysis of different rice genomes, and the hypothesis was put forward that such duplications might arise due to the concerted repair of adjacent single-strand breaks (SSBs). Applying the CRISPR/Cas9 technology, we were able to test this hypothesis experimentally in the model plant Arabidopsis thaliana. Using a Cas9 nickase to induce adjacent genomic SSBs in different regions of the genome (genic, intergenic, and heterochromatic) and at different distances (∼20, 50, and 100 bps), we analyzed the repair outcomes by deep sequencing. In addition to deletions, we regularly detected the formation of direct repeats close to the break sites, independent of the genomic context. The formation of these duplications as well as deletions may be associated with the presence of microhomologies. Most interestingly, we found that even the induction of two SSBs on the same DNA strand can cause genome alterations, albeit at a much lower level. Because such a scenario reflects a natural step during nucleotide excision repair, and given that the germline is set aside only late during development in plants, the repair of adjacent SSBs indeed seems to have an important influence on the shaping of plant genomes during evolution. T he opportunity to experimentally investigate the outcome of double-strand break (DSB) repair in eukaryotic organisms was enabled by the use of site-specific endonucleases targeting unique genomic sequences. Early work using the meganuclease I-SceI more than 20 y ago demonstrated that the induction of a DSB leads to enhanced homologous recombination (HR) in multicellular eukaryotes (1). This finding led to the application of I-SceI as a tool for detailed analysis of genomic changes that might occur due to DSB repair via nonhomologous end-joining (NHEJ) in plants. Thus, it was possible to demonstrate that along with inducing various kinds of deletions (2, 3), DSB repair also can be associated with the integration of T-DNA, as well as with the copying of genomic sequences into the break (4, 5).Recently, a novel type of programmable nuclease was introduced by the discovery of the molecular mechanism of the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) system (6, 7). In this system, the endonuclease Cas9 is guided by two specialized RNAs (CRISPR RNA and transactivating CRISPR RNA) via direct base-pairing to bind and cleave invading harmful DNA. It has been demonstrated in vitro and in vivo that by fusing the two RNAs to a so-called singleguide RNA (sgRNA), the system can serve as a highly efficient and precisely programmable nuclease for genome eng...

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Section: The Role Of Paired Ssbs On Opposite Strands In the Formation Ofmentioning

confidence: 99%

Repair of adjacent single-strand breaks is often accompanied by the formation of tandem sequence duplications in plant genomes

Schiml

Fauser

Puchta

2016

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

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“…In these studies, a high-throughput sequencing method, known as excision repair-sequencing (XR-seq), was used to analyze oligonucleotide fragments excised during NER of CPDs or 6-4PPs. These studies found increased repair activity associated with TC-NER of the TS of actively expressed genes and noncoding transcripts (7,8). There was also more rapid repair in active chromatin domains (i.e., enhancers and promoters) and DNase I hypersensitivity regions, and slower repair associated with repressed and heterochromatin regions (8).…”

mentioning

confidence: 95%

“…These studies found increased repair activity associated with TC-NER of the TS of actively expressed genes and noncoding transcripts (7,8). There was also more rapid repair in active chromatin domains (i.e., enhancers and promoters) and DNase I hypersensitivity regions, and slower repair associated with repressed and heterochromatin regions (8). Reanalysis of these data by other groups indicated that there is decreased repair activity associated with transcription factor binding sites (9) and transcription initiation sites (10), suggesting that NER may be inhibited by the binding of transcription factors and the transcription machinery to DNA.…”

mentioning

confidence: 97%

“…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 activity in UV-irradiated human fibroblasts (7,8). In these studies, a high-throughput sequencing method, known as excision repair-sequencing (XR-seq), was used to analyze oligonucleotide fragments excised during NER of CPDs or 6-4PPs.…”

mentioning

confidence: 99%

“…Our understanding of how NER operates in different sequence and chromatin contexts has been aided by two recent genome-wide surveys of NER activity in UV-irradiated human fibroblasts (7,8). In these studies, a high-throughput sequencing method, known as excision repair-sequencing (XR-seq), was used to analyze oligonucleotide fragments excised during NER of CPDs or 6-4PPs.…”

mentioning

confidence: 99%

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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.

140

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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Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses

et al. 2021

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Mitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. We employ a molecular approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe (UBER) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.

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

Cited by 160 publications

References 44 publications

Repair of adjacent single-strand breaks is often accompanied by the formation of tandem sequence duplications in plant genomes

Repair of adjacent single-strand breaks is often accompanied by the formation of tandem sequence duplications in plant genomes

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

Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses

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