Cisplatin DNA damage and repair maps of the human genome at single-nucleotide resolution

Abstract

Cisplatin is a major anticancer drug that kills cancer cells by damaging their DNA. Cancer cells cope with the drug by removal of the damages with nucleotide excision repair. We have developed methods to measure cisplatin adduct formation and its repair at single-nucleotide resolution. "Damage-seq" relies on the replication-blocking properties of the bulky base lesions to precisely map their location. "XR-seq" independently maps the removal of these damages by capturing and sequencing the excised oligomer released during repair. The damage and repair maps we generated reveal that damage distribution is essentially uniform and is dictated mostly by the underlying sequence. In contrast, cisplatin repair is heterogeneous in the genome and is affected by multiple factors including transcription and chromatin states. Thus, the overall effect of damages in the genome is primarily driven not by damage formation but by the repair efficiency. The combination of the Damageseq and XR-seq methods has the potential for developing novel cancer therapeutic strategies.] is a major frontline drug in the treatment of lung, colorectal, ovarian, and head-and-neck cancers (1) and has been used in the clinic since 1978. It kills cancer cells by damaging their DNA, mainly by forming Pt-d(GpG) and, to a lesser extent, Pt-d(ApG), Pt-d(GpXpG) intrastrand diadduct, and at lower frequency, Pt-G-G interstrand cross-links (2-4). The cisplatin-induced damage is repaired by nucleotide excision repair (5-8). In excision repair, the damage in DNA is excised from the genome as a single-stranded oligomer of ∼30 nt. The single-stranded gap in the genome is filled in by DNA polymerases and ligated, resulting in error-free repair. Although cisplatin is effective in the treatment of the indicated types of cancers, in some of the cases, drug resistance is observed. The cause of the resistance is multifactorial, and DNA repair is considered to be one of the contributing factors, although the degree to which DNA repair contributes to resistance is still unclear. Here, we describe the development of methods to measure cisplatin adduct formation and repair at single-nucleotide resolution. Using these methods, named "Damage-seq" and "eXcision Repairseq" (XR-seq), we have generated single-nucleotide resolution maps for both the damage induced by cisplatin and its removal by nucleotide excision repair (9, 10). Our method should be applicable for studying drug resistance and for optimization of cancer chemotherapy regimens. ResultsIn this work, we present the cisplatin damage and repair maps for the entire genome of the human lymphocyte cell line GM12878. This cell line was chosen because it has been extensively characterized by the ENCODE project (11). Similar experiments with oxaliplatin, a third generation derivative of cisplatin, yielded similar results and are presented in SI Appendix, SI Materials and Methods. All experiments were performed with two biological replicates.Cisplatin DNA Damage Map. We developed the method of Damage-seq based on the fact th...

Photodamage repair pathways contribute to the accurate maintenance of the DNA methylome landscape upon UV exposure

“…It was also demonstrated that a positive correlation exists between DNA methylation patterns and mutations induced by UV irradiation (Willing et al, 2016). In addition, UV-C damages both euchromatin and heterochromatin whilst other genotoxic agents inducing bulky DNA lesions, like cisplatin, acts mostly in euchromatin (Hu et al, 2016). Therefore, all these parameters allow considering that, in addition to the presence of di-pyrimidines, DNA methylation, nucleosome density and histone variants/PTM may synergistically contribute to favor photolesions formation in heterochromatin vs euchromatin.…”

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

“…Based on DNA sequence elements and histone posttranslational modifications, 15 chromatin states have been defined (35). In previous studies, we found that although these states did not affect cisplatin-induced damage formation, they did affect repair efficiencies, particularly in active promoters, enhancers, and transcribed gene bodies (10,13). We analyzed all 15 chromatin states for CPD, BPDE-dG, and (6-4)PP repair (Fig.…”

“…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)(11)(12).…”

NEK5 interacts with topoisomerase IIβ and is involved in the DNA damage response induced by etoposide

“…2 The understanding of DNA damage response (DDR) and repair allowed the development of chemotherapeutical drugs to affect and defeat cancer cells, such as cisplatin and etoposide. 3,4 The different protein kinases related to the regulation of DDR and repair rely on a class of protein kinases: the NIMA-related kinases (NEKs) family. In general, NEKs have a conserved N-terminal catalytic domain, with approximately 40% amino-acid similarity, and a regulatory domain, which contributes to the uniqueness of each NEK protein.…”