An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells

Choi, Jun Hyuk; Kim, So Young; Kim, Sook Kyung; Kemp, Michael G.; Sancar, Aziz

doi:10.1074/jbc.m115.690354

Cited by 32 publications

(42 citation statements)

References 41 publications

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“…However, there is currently no in vitro excision repair assay for plant nucleotide excision repair. We therefore adapted the in vivo excision assay that we recently developed in our laboratory for studying nucleotide excision repair in mammalian cells in vivo (12)(13)(14). In this assay, cells are lysed at various time points following UV irradiation and the excised oligonucleotide products of nucleotide excision repair are isolated by immunoprecipitation with antibodies against either the repair factor TFIIH (because the excised oligonucleotide is released in a complex with the core repair factor TFIIH) or against specific UV photoproducts (12, 20).…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, there have been no mechanistic studies on plant nucleotide excision repair, although it is known that plants can remove cyclobutane pyrimidine dimers (CPDs) and pyrimidine pyrimidone photoproducts [(6-4)PPs] in a photolyase-independent manner (6,10,11), presumably by nucleotide excision repair. Here, we have used an Arabidopsis cell line and the in vivo excision assay recently developed in our laboratory (12)(13)(14) to demonstrate that Arabidopsis removes these photoproducts by dual incisions in a manner that is virtually identical to human nucleotide excision repair.…”

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Nucleotide excision repair by dual incisions in plants

Cantürk

Karaman

Selby

et al. 2016

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

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Plants use light for photosynthesis and for various signaling purposes. The UV wavelengths in sunlight also introduce DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] that must be repaired for the survival of the plant. Genome sequencing has revealed the presence of genes for both CPD and (6-4)PP photolyases, as well as genes for nucleotide excision repair in plants, such as Arabidopsis and rice. Plant photolyases have been purified, characterized, and have been shown to play an important role in plant survival. In contrast, even though nucleotide excision repair gene homologs have been found in plants, the mechanism of nucleotide excision repair has not been investigated. Here we used the in vivo excision repair assay developed in our laboratory to demonstrate that Arabidopsis removes CPDs and (6-4)PPs by a dual-incision mechanism that is essentially identical to the mechanism of dual incisions in humans and other eukaryotes, in which oligonucleotides with a mean length of 26-27 nucleotides are removed by incising ∼20 phosphodiester bonds 5′ and 5 phosphodiester bonds 3′ to the photoproduct.lants and other organisms that depend on photosynthesis are, by necessity, exposed to more sunlight than other organisms that are chemotrophs or heterotrophs. Hence, plants are expected to receive more exposure to UV wavelengths of light than other organisms. The genotoxic effects of UV are somewhat mitigated by the reflection of UV by the waxy leaf surface and absorbance of UV by the intracellular pigments that are present at high concentration in plant cells, including carotenoids and flavonoids. Nevertheless, plants still receive considerable amounts of DNA-damaging UV radiation and therefore must have the means to cope with the damage to ensure their survival. Indeed, DNA sequencing has revealed that plant genomes contain genes that are homologous to the genes of all major DNA repair pathways, including photoreactivation, nucleotide excision repair, base excision repair, and recombination/double-strand break repair (1-6).However, biochemical studies of these DNA repair mechanisms have been limited. Of significance, Arabidopsis photolyases have been expressed in heterologous systems, purified, and characterized (7-9). Similarly, some of the enzymes of the base excision repair and recombination/double-strand break repair systems have been studied. In contrast, there have been no mechanistic studies on plant nucleotide excision repair, although it is known that plants can remove cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)PPs] in a photolyase-independent manner (6, 10, 11), presumably by nucleotide excision repair. Here, we have used an Arabidopsis cell line and the in vivo excision assay recently developed in our laboratory (12)(13)(14) to demonstrate that Arabidopsis removes these photoproducts by dual incisions in a manner that is virtually identical to human nucleotide excision repair. ResultsNucleotide Ex...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Nucleotide excision repair by dual incisions in plants

Cantürk

Karaman

Selby

et al. 2016

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

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“…Thus, the presence of DNA oligonucleotides of this length in complex with TFIIH is a defining feature of the human nucleotide excision repair system and can be used to monitor excision repair in cultured cells. Moreover, we have shown that this general methodology is useful for monitoring excision repair kinetics in response to a wide variety of DNA-damaging agents that generate DNA adducts that are targeted by the nucleotide excision repair system (35).…”

Section: Resultsmentioning

confidence: 99%

Analysis of Ribonucleotide Removal from DNA by Human Nucleotide Excision Repair

Lindsey-Boltz

Kemp

et al. 2015

Journal of Biological Chemistry

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Ribonucleotides are incorporated into the genome during DNA replication. The enzyme RNase H2 plays a critical role in targeting the removal of these ribonucleotides from DNA, and defects in RNase H2 activity are associated with both genomic instability and the human autoimmune/inflammatory disorder Aicardi-Goutières syndrome. Whether additional general DNA repair mechanisms contribute to ribonucleotide removal from DNA in human cells is not known. Because of its ability to act on a wide variety of substrates, we examined a potential role for canonical nucleotide excision repair in the removal of ribonucleotides from DNA. However, using highly sensitive dual incision/excision assays, we find that ribonucleotides are not efficiently targeted by the human nucleotide excision repair system in vitro or in cultured human cells. These results suggest that nucleotide excision repair is unlikely to play a major role in the cellular response to ribonucleotide incorporation in genomic DNA in human cells.

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“…BPDE preferentially forms bulky covalent DNA adducts at N2 position of guanines and causes mutations if these BPDE-deoxyguanosines (BPDE-dGs) are not efficiently eliminated by nucleotide excision repair (3). Various methods of varying resolutions have been developed for mapping DNA damage and repair genome-wide (4)(5)(6)(7)(8)(9). We previously reported a method, termed excision repair-sequencing (XR-seq), for mapping nucleotide excision repair (6).…”

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Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene

Adebali

et al. 2017

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

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Benzo [a]pyrene (BaP), a polycyclic aromatic hydrocarbon, is the major cause of lung cancer. BaP forms covalent DNA adducts after metabolic activation and induces mutations. We have developed a method for capturing oligonucleotides carrying bulky base adducts, including UV-induced cyclobutane pyrimidine dimers (CPDs) and BaP diol epoxide-deoxyguanosine (BPDE-dG), which are removed from the genome by nucleotide excision repair. The isolated oligonucleotides are ligated to adaptors, and after damage-specific immunoprecipitation, the adaptor-ligated oligonucleotides are converted to dsDNA with an appropriate translesion DNA synthesis (TLS) polymerase, followed by PCR amplification and next-generation sequencing (NGS) to generate genome-wide repair maps. We have termed this method translesion excision repair-sequencing (tXR-seq). In contrast to our previously described XR-seq method, tXR-seq does not depend on repair/removal of the damage in the excised oligonucleotides, and thus it is applicable to essentially all DNA damages processed by nucleotide excision repair. Here we present the excision repair maps for CPDs and BPDE-dG adducts generated by tXR-Seq for the human genome. In addition, we report the sequence specificity of BPDE-dG excision repair using tXR-seq. N ucleotide excision repair is a versatile repair pathway that removes a variety of DNA damages, including UV-and benzo[a]pyrene (BaP)-induced DNA damages. BaP, a widespread carcinogen, is the major cause of lung cancer (1). It is produced by incomplete combustion of organic materials and converted to the ultimate mutagen, BaP diol epoxide (BPDE), through enzymatic metabolism (2). BPDE preferentially forms bulky covalent DNA adducts at N2 position of guanines and causes mutations if these BPDE-deoxyguanosines (BPDE-dGs) are not efficiently eliminated by nucleotide excision repair (3). Various methods of varying resolutions have been developed for mapping DNA damage and repair genome-wide (4-9). We previously reported a method, termed excision repair-sequencing (XR-seq), for mapping nucleotide excision repair (6). This method has been used to generate excision repair maps for UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4)pyrimidine-pyrimidone photoproducts [(6-4)PPs], as well as cisplatin and oxaliplatin-induced Pt-d(GpG) diadducts for the human genome and CPDs for the Escherichia coli genome (10-12).Although the XR-seq method has been quite useful in determining the effects of various factors, such as genetic background, transcription, posttranscriptional histone modification, and chromatin states, on the timing and efficiency of repair (13), the method in its original form requires the reversal of damage in the excised oligonucleotide (26-27 mers in humans and 12-13 mers in E. coli) by enzymatic or chemical means before it can be processed for nextgeneration sequencing (NGS) and generation of repair maps. As such, this method has limitations, because it is not possible to reverse, either enzymatically or chemically, most DNA lesions removed f...

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An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells

Cited by 32 publications

References 41 publications

Nucleotide excision repair by dual incisions in plants

Nucleotide excision repair by dual incisions in plants

Analysis of Ribonucleotide Removal from DNA by Human Nucleotide Excision Repair

Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene

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