Recent Advances in DNA Lesion Mapping and Repair Mechanisms.

Recognition and repair of DNA lesions are critical for cell survival. Herein, we highlight recent advances in the sequencing, repair mechanisms, and biological consequences of DNA lesions presented at the 2022 Fall American Chemical Society meeting.

Development of Comprehensive Ultraperformance Liquid Chromatography-High-Resolution Mass Spectrometry Assays to Quantitate Cisplatin-Induced DNA-DNA Cross-Links.

Cisplatin (CP) is a common antitumor drug that is used to treat many solid tumors. The activity of CP is attributed to the formation of DNA-DNA cross-links, which consist of 1,2-intra-, 1,3-intra-, and interstrand cross-links. To better understand how each intrastrand cross-link contributes to the activity of CP, we have developed comprehensive ultraperformance liquid chromatography-selective ion monitoring (UPLC-SIM) assays to quantify 1,2-GG-, 1,2-AG-, 1,3-GCG-, and 1,3-GTG-intrastrand cross-links. The limit of quantitation for the developed assays ranged from 5 to 50 fmol or as low as 6 cross-links per 108 nucleotides. To demonstrate the utility of the UPLC-SIM assays, we first performed in vitro cross-link formation kinetics experiments. We confirmed that the 1,2-GG-intrastrand cross-links were the most abundant intrastrand cross-link and formed at a faster rate compared to 1,2-AG- and 1,3-intrastrand cross-links. Furthermore, we investigated the repair kinetics of intrastrand cross-links in CP-treated wild-type and nucleotide excision repair (NER)-deficient U2OS cells. We observed a slow decrease of both 1,2- and 1,3-intrastrand cross-links in wild-type cells and no evidence of direct repair in the NER-deficient cells. Taken together, we have demonstrated that our assays are capable of accurately quantifying intrastrand cross-links in CP-treated samples and can be utilized to better understand the activity of CP.

New Synthetic Analogs of Nitrogen Mustard DNA Interstrand Cross-Links and Their Use to Study Lesion Bypass by DNA Polymerases.

Nitrogen mustards are a widely used class of antitumor agents that exert their cytotoxic effects through the formation of DNA interstrand cross-links (ICLs). Despite being among the first antitumor agents used, the biological responses to NM ICLs remain only partially understood. We have previously reported the generation of NM ICL mimics by incorporation of ICL precursors into DNA using solid-phase synthesis at defined positions, followed by a double reductive amination reaction. However, the structure of these mimics deviated from the native NM ICLs. Using further development of our approach, we report a new class of NM ICL mimics that only differ from their native counterpart by substitution of dG with 7-deaza-dG at the ICL. Importantly, this approach allows for the synthesis of diverse NM ICLs, illustrated here with a mimic of the adduct formed by chlorambucil. We used the newly generated ICLs in reactions with replicative and translesion synthesis DNA polymerase to demonstrate their stability and utility for functional studies. These new NM ICLs will allow for the further characterization of the biological responses to this important class of antitumor agents.

N6-(2-Deoxy-d- erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5- N-(2-hydroxy-3-buten-1-yl)-formamidopyrimidine Adducts of 1,3-Butadiene: Synthesis, Structural Identification, and Detection in Human Cells.

1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. BD is metabolically activated by cytochrome P450 monooxygenases to 3,4-epoxy-1-butene (EB), which alkylates DNA to form a range of nucleobase adducts. Among these, the most abundant are the hydrolytically labile N7-guanine adducts such as N7-(2-hydroxy-3-buten-1-yl)-guanine (N7-EB-dG). We now report that N7-EB-dG can be converted to the corresponding ring open N6-(2-deoxy-d- erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5- N-(2-hydroxy-3-buten-1-yl)-formamidopyrimidine (EB-Fapy-dG) adducts. EB-Fapy-dG lesions were detected in EB-treated calf thymus DNA and in EB-treated mammalian cells using quantitative isotope dilution nanoLC-ESI+-MS/MS. EB-Fapy-dG adduct formation in EB-treated calf thymus DNA was concentration dependent and was greatly accelerated at an increased pH. EB-FAPy-dG adduct amounts were 2-fold higher in base excision repair-deficient NEIL1-/- mouse embryonic fibroblasts (MEF) as compared to isogenic controls (NEIL1+/+), suggesting that this lesion may be a substrate for NEIL1. Furthermore, NEIL1-/- cells were sensitized to EB treatment as compared to NEIL1+/+ fibroblasts. Overall, our results indicate that ring-opened EB-FAPy-dG adducts form under physiological conditions, prompting future studies to determine their contributions to genotoxicity and mutagenicity of BD.

Cross-linking of the DNA repair protein O6-alkylguanine DNA alkyltransferase to DNA in the presence of cisplatin.

1,1,2,2-cis-diamminedichloroplatinum (II) (cisplatin) is a chemotherapeutic agent widely used in the clinic to treat various cancers. The antitumor activity of cisplatin is generally attributed to its ability to form intrastrand and interstrand DNA-DNA cross-links via sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also induces DNA- protein lesions (DPCs) that may contribute to its biological effects due to their ability to block DNA replication and transcription. We previously reported that over 250 nuclear proteins including high mobility group proteins, histone proteins, and elongation factors formed DPCs in human HT1080 cells treated with cisplatin (Ming et al. Chem. Res. Toxicol. 2017, 30, 980-995). Interestingly, cisplatin-induced DNA-protein conjugates were reversed upon heating, by an unknown mechanism. In the present work, DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate the molecular details of cisplatin-mediated DNA-protein cross-linking and to establish the mechanism of their reversal. We found that AGT is readily cross-linked to DNA in the presence of cisplatin. HPLC-ESI+-MS/MS sequencing of tryptic peptides originating from dG-Pt-AGT complexes revealed that the cross-linking occurred at six sites within this protein including Glu110, Lys125, Cys145, His146, Arg147, and Cys150. Cisplatin-induced Lys-Gua cross-links (1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) were detected by HPLC-ESI+-MS/MS of total digests of modified protein in comparison with the corresponding authentic standard. Upon heating, dG-Pt-AGT complexes were subject to platination migration from protein to DNA, forming cis-[Pt(NH3)2{d(GpG)}] cross-links which were detected by HPLC-ESI+-MS/MS. Our results provide a new insight into the mechanism of cisplatin-mediated DNA-protein cross-linking and their dynamic equilibrium with the corresponding DNA-DNA lesions.