Mass Spectrometry-Based Characterization, Quantitation, And Repair Investigations Of Complex DNA Lesions

University of Minnesota Ph.D. dissertation. March 2018. Major: Medicinal Chemistry. Advisor: Natalia Tretyakova. 1 computer file (PDF); xxv, 359 pages.DNA is constantly under the threat of damage by various endogenous and exogenous agents, leading to the structural modification of nucleobases (DNA adducts). These DNA adducts can range from smaller nucleoside monoadducts and exocyclic adducts, to the helix distorting and super-bulky DNA-DNA cross-links and DNA-protein cross-links. If not repaired, DNA adducts can inhibit crucial biological processes such as DNA replication, leading to adverse consequences such as mutagenesis and carcinogenesis. Therefore, understanding the atomic connectivity, extent of formation, and repair of DNA adducts is crucial to fully elucidating the biological consequences of the adduct. DNA-protein cross-links (DPCs) are ubiquitous, super-bulky DNA lesions that form when proteins become irreversibly trapped on chromosomal DNA. The structural complexity of cross-linking and the diversity of proteins susceptible to DPC formation represents significant challenges to studying the biological consequence of these adducts. In the first part of the thesis, we identified the protein constituents, structural characterized and quantified, and investigated the repair mechanism of bis-electrophile (Chapter 2) and reactive oxygen species (ROS, Chapters 3 and 4)-induced DPCs. In Chapter 2, we investigated DPC formation after exposure to N,N-bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and N,N-bis-(2-chloroethyl)-ethylamine (nornitrogen mustard, NOR), the two biologically active metabolites of the antitumor agent cyclophosphamide. A mass spectrometry-based proteomics approach was employed to characterize the protein constituents of PM- and NOR-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells. HPLC-ESI+-MS/MS analysis of proteolytic digests of DPC-containing DNA from NOR-treated cells revealed a concentration-dependent formation of N-[2-[cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]amine (Cys-NOR-N7G) conjugates, confirming that it cross-links cysteine thiols of proteins to the N-7 position of guanines in DNA. A sensitive and accurate Cys-NOR-N7G isotope dilution tandem mass spectrometry assay was developed to quantify PM-induced DPC formation and repair in mammalian cells proficient or deficient in a DNA repair pathway. In Chapters 3, we employed the model of left anterior descending artery ligation/reperfusion surgery in rat to show that ischemia/reperfusion injury is associated with the formation of hydroxyl radical-induced DNA-protein cross-links (DPCs) in cardiomyocytes. Mass spectrometry based experiments revealed that these conjugates were formed by a free radical mechanism and involved thymidine residues of DNA and tyrosine side chains of proteins (dT-Tyr). Quantitative proteomics experiments utilizing Tandem mass tags (TMT) revealed that radical-induced DPC formation increase after LAD-ligation/reperfusion compared to the control sham surgery. Using the developed dT-Tyr nanoLC-ESI+-MS/MS assay, we investigated the role of the metalloprotease Spartan (SPRTN) in the repair of radical-induced DPCs (Chapter 4). Analysis of the brain, liver, heart, and kidneys of wild type (SPRTN+/+) and hypomorphic (SPRTN f/-) mice revealed a 1.5 – 2-fold increase in dT-Tyr in the hypomorphic mice, providing direct evidence that Spartan plays a role in the repair of radical-induced DPCs. Finally, we investigated the formation of formamidopyrimidine (FAPy) adducts after exposure to 3,4-epoxybutene, an epoxide metabolite of the known carcinogen 1,3-butadiene (Chapter 5). We successfully synthesized and structurally characterized a novel BD-induced DNA adduct EB-FAPy-dG, and developed a sensitive isotope dilution tandem mass spectrometry assay for its detection in vitro and in cells. To our knowledge, this is the first report of a BD-induced FAPy adduct, and future studies will examine whether BD-induced FAPy adducts In summary, during the course of this Thesis, we utilized mass spectrometry-based proteomics techniques to identify the proteins susceptible to PM- and ROS-induced DPC formation. After structurally characterizing the atomic connectivity of these adduces, we developed sensitive and accurate isotope dilution tandem mass spectrometry assays to perform absolute quantitation of PM- and ROS-induced DPC formation in cells and tissues. These assays were further utilized to begin investigating the repair mechanism of DPCs in cells and tissues, including providing direct evidence that the metalloprotease Spartan is involved in the repair of radical-induced DPCs. Finally, we detected EB-FAPy-dG formation in vitro and in vivo, the first evidence of 1,3-butadiene induced formamidopyrimidine formation

HDM2 ERKs PCNA

In this issue, a study by Groehler and Lannigan (2010. J. Cell Biol. doi:10.1083/jcb.201002124) sheds light on the regulation of proliferating cell nuclear antigen (PCNA) turnover and how it is counteracted by the small chromatin-bound kinase ERK8 (extracellular signal-regulated kinase 8). Importantly, inactivation of ERK8 results in genome instability and is associated with cell transformation

A chromatin-bound kinase, ERK8, protects genomic integrity by inhibiting HDM2-mediated degradation of the DNA clamp PCNA

ERK8 prevents genome instability by blocking the association of the HDM2 E3 ubiquitin ligase with the genome-protecting protein PCNA

Cooperative Marketing

Published by the students of the College of Agriculture, University of Arizona.This article is part of the Arizona Agriculturist collections. Digital access to this material is made possible by the University of Arizona Libraries. Permission to use or to order reproductions must be obtained from the University of Arizona Libraries, Special Collections. Contact us at [email protected], or (520) 621-6423

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. © Chemical Research in Toxicology. All rights reserved.11Nsciescopu

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

Assessment of Musculoskeletal Pain and Physical Demands Using a Wearable Smartwatch Heart Monitor among Precast Concrete Construction Workers: A Field Case Study

This study aimed to quantify musculoskeletal symptoms/pain and characterize the physical demands at work and outside of work among precast concrete workers. Direct heart rate (HR) measurements and self-reported activity levels were used to estimate the physical demands. A total of 27 precast construction workers participated in a survey, and 21 wore a HR monitor smart watch for seven days. The HR data were parsed in minutes associated with occupational and nonoccupational physical activity. Correlation analysis and multivariate regression models were conducted to assess the associations between direct measured physical activity with self-reported physical activity, body mass index (BMI), years of work, smoking, and Borg ratings of perceived exertion (RPE). Approximately half of the participants experienced musculoskeletal symptoms in the last seven days and moderate functional limitations in carrying out activities of daily living (ADLs). The regression model revealed a positive relationship between direct measured moderate occupational physical activity (OPA) and the Borg RPE. Furthermore, an inverse association was found with BMI, smoking status, and years of work. The workers accrued a median of 415 min of moderate OPA per week. The findings showed a high amount of moderate OPA minutes per week and musculoskeletal issues among the precast concrete workers