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

Abstract

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...

Repair-Resistant DNA Lesions

“…23,26,30 While the thymine dimer T^T CPD opposite its canonical adenine (AA) bases in the complementary strand is NER-resistant, it becomes an excellent substrate of NER when these AA bases are replaced by “mismatched” GG. 10 The development of genome-wide methods of analysis of the formation and repair of these UV photolesions in human fibroblasts, 31 human lymphocytes, 32 and yeast 30 genomes has provided novel insights into the base sequence dependence of mutational processes at the single nucleotide level of resolution.…”

“…32 This approach is based on the detection of DNA adducts that are substrates of NER in cellular environments. A sequencing library for tXR-seq analysis was generated by the immunoprecipitation of the intermediate NER dual excision products complexed with TFIIH, followed by the construction of a suitable library for next generation sequencing.…”

Do the Number of Cigarettes Smokes per day Contribute to the Incident of Malignant Cancer?

“…It happens due to the accumulation of dangerous chemicals in cigarettes which are able to divide the DNA in cells into abnormalities in all part of bodies (Morris et al, 1997;Valavanidis et al, 2009;Moktar et al, 2011). Moreover, at least 60 chemicals in cigarette are carcinogens including arsenic as a major cause of skin and liver cancer and benzoapyrene as a major cause of lung cancer and oral cavity cancer (Valavanidis et al, 2009;Li et al, 2017). Praud et al, (2018) stated that the risk of gastric cancer depends on dose and duration of smoking, and the decrease in risk following stopping smoking.…”

“…There are at least 17 types incident of cancer including larynx, lung, hypo pharynx, nasal cavities, bladder, oesophagus and liver due to cigarette smoking behaviour (Baade, 2015;Alexandrov, 2016). The dangerous chemicals in the cigarette smoked might change the DNA, triggering abnormalities cell, containing free radicals, carcinogenic and binding the blood fat are able to grow the cell of cancer (Gandini et al, 2008;Valavanidis et al, 2009;Moktar et al, 2011;Li et al, 2017). However, the manifestation of malignancy cancer needs a long period, which usually appear after 5-20 years (Hansen et al, 2013;Furrukh, 2013).…”

Nucleotide excision repair capacity increases during differentiation of human embryonic carcinoma cells into neurons and muscle cells

“…In our studies, we monitored the repair rates of UV damage at various stages of differentiation and in two distinct differentiation lineages. With the advancement of high-throughput sequencing-based methods for mapping of DNA damage and repair (45)(46)(47)(48)(49)(50), the nucleotide-resolution genome-wide DNA damage formation and repair profiles in stem cells and differentiated cells can be achieved and analyzed in future studies to better understand the molecular mechanisms underlying the dynamics of nucleotide excision repair capacity over differentiation process.…”