Browsing by Subject "DNA-protein cross-links"

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    Biological Consequences Of Complex Dna Lesions Induced By Bis-Electrophiles
    (2014-08) Wickramaratne, Susith
    Genomic DNA is constantly modified by bis–electrophiles which induce a wide array of DNA adducts. DNA adducts can threaten cell viability and genomic integrity by interfering with DNA replication and transcription. Unless repaired, they can be misread by DNA polymerases, leading to heritable mutations and cancer. Amongst known human carcinogens is 1,3–butadiene (BD), an important industrial chemical and environmental pollutant. BD is metabolized to reactive epoxides that induce a range of DNA adducts. In the first part of this thesis, we investigated the ability of human cells to repair three potentially mutagenic BD–adenine lesions: N 6–(2–hydroxy–3–buten–1–yl)–2 ’–deoxyadenosine, 1,N 6–(2–hydroxy–3–hydroxymethylpropan–1,3–diyl)–2 ’–deoxyadenosine and N 6–,N 6–(2,3–dihydroxybutan–1,4–diyl)–2 ’–deoxyadenosine. Repair assays using site– and stereo–specifically adducted oligodeoxynucleotides revealed that all three BD–dA adducts were recognized by base excision repair (BER) pathway. Repair inhibition by a BER inhibitor, and 5’ excision products characterized by HPLC–ESI––MS/MS analysis confirmed the involvement of BER. Exposure to bis–electrophiles or reactive oxygen species can irreversibly trap cellular proteins on DNA, forming super–bulky lesions known as DNA–protein cross–links (DPCs). The complexity and diversity of DPCs have prevented researchers from investigating their biological effects. In the second part of this thesis, we have developed bioconjugation methods to generate site–specific DPCs and examined their effects on DNA replication. Hydrolytically stable 7–deazaguanine adducted DPCs, which are structurally analogous to cellular DPCs induced by antitumor nitrogen mustards, were prepared by post–synthetic reductive amination. Further, Huisgen cycloaddition was used to generate DPCs involving the C5–thymidine of DNA. Replication bypass using human translesion synthesis polymerases η and κ revealed that large polypeptides cross–linked to either 7–deazaguanine or C5–thymidine completely blocked replication, while the corresponding decapeptide cross–links were bypassed, suggesting that bypass of DPCs in cells is mediated by proteolytic degradation of the cross–linked proteins. Steady–state kinetic studies provided evidence for the highly error–prone bypass of C5–thymidine peptide cross–links and error–free bypass of N7–guanine cross–linked peptides. Further, HPLC–ESI––MS/MS characterization of replication products of C5–thymidine peptide cross–links revealed large numbers of deletion and substitution mutations. Taken together, our data suggest that the efficiency and the fidelity of DNA replication past DPCs are dependent on the lesion size, the cross–linking site and DNA polymerase identity.
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    Chemistry and Biology of DNA-protein cross-links
    (2019-02) Ji, Shaofei
    DNA-protein cross-links (DPCs) are ubiquitous DNA lesions formed when proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. Because of their considerable size, DPCs interfere with the progression of replication and transcription machineries, potentially contributing to mutagenesis and carcinogenesis. However, unlike small DNA lesions of which the biological consequences and repair mechanisms have been well characterized, biological effects and repair mechanism of DPC lesions remain to be established. A significant challenge in the field is the structural diversity of DPC lesions and the scarcity of experimental mythologies to create site-specific DNA-protein conjugates. The main objective of this thesis was to synthesize model DPC and to investigate their biological consequences and repair mechanisms. In Chapter II, we discovered, characterized and quantified 5-formylcytosine(5fC) mediated DNA-histone conjugates in human cells. 5-Formylcytosine (5fC) is an endogenous DNA modification enzymatically generated in the genome as an oxidation product of 5-methyl-dC (5mC). While 5mC is known to be an epigenetic mark that controls the levels of gene expression, the biological functions of 5fC are incompletely understood. In this chapter, we discovered that 5fC bases in DNA readily form Schiff base conjugates with Lys side chains of nuclear proteins such as histones, forming covalent DNA-protein conjugates. Isotope dilution nanoLC-ESI-MS/MS methodology was employed to detect and quantify 5fC-lys conjugate in human cells. We hypothesize that reversible 5fC-histone cross-linking contributes to epigenetic signaling, transcriptional regulations and chromatin remodeling. After the discovery of 5fC-mediated DNA-histone crosslinks in mammalian cells, we investigated their effects on DNA replication in Chapter III. DNA substrates containing site-specific DPCs were subjected to in vitro translesion synthesis (TLS) in the presence of TLS DNA polymerases. We found that DPCs containing various full-length proteins conjugated to DNA via the C-5 position of cytosine completely blocked human DNA polymerases, while the corresponding lesions containing shorter peptides were bypassed by translesion synthesis (TLS) polymerases. These results are consistent with the proposed DPC repair pathway in the literature, in which full-length DPCs are subjected to proteolytic degradation to generate short DNA-peptide cross-links, which may serve as substrates for translesion synthesis. In addition, our steady-state kinetics analysis and mass- spectrometry-based sequencing and quantification revealed that the bypass of DNA- peptide cross-links by human TLS polymerases was highly error-prone, introducing significant amounts of C to T and deletion mutations. In Chapter IV, we investigated the effects of DPCs on transcription using two model DPCs where the proteins are conjugated to the C5 position of cytosine or the C7 position of 7-deazaguanine. The latter serves as a hydrolytically stable model of the N7- guanine lesions, which commonly form upon exposure to bis-electrophiles such antitumor agents. We found that full-length proteins cross-linked to either 5fC or 7-deazaguanine completely blocked T7 RNA polymerase, while relatively short peptide cross-links were bypassed, although with low efficiency. Interestingly, the two model DPCs exhibited completely different mutagenic patterns are revealed by PCR and mass spectrometry based assays. While the bypass of peptide cross-linked to 7-deaza-G by T7 RNA polymerases induced very small numbers of mutations, transcription past peptide lesions conjugated to C-5 of C induced significant amounts of C to T transcriptional mutations. In Chapter V, we investigated the effects of 5fC-mediated DNA-peptide/protein cross-links on transcription and its potential repair by nucleotide excision repair (NER) in living cells. To accomplish this goal, structurally defined DPCs were site-specifically incorporated into plasmid molecules, which was then transfected into wild type cells or cells deficient in NER. RT-PCR and LC-MS/MS based strategy was then employed to quantitatively study the effects of DPC lesions on efficiency and fidelity of transcription in mammalian cells. We found that the presence of peptide cross-links conjugated to C-5 of cytosine significantly inhibited DNA transcription in human embryonic kidney cells. However, in contrast to our in vitro results, no transcriptional mutagenesis was observed. In addition, we compared the transcription bypass efficiencies of DpC lesions in wild-type and NER-deficient cell-lines, and also conducted the in vitro NER assays using cell-free extracts from human HeLa cells. Collectively, our data suggested that 5fC-mediated DNA- peptide cross-links are poor NER substrates, requiring a different pathway for their repair. Recent studies suggested that the bulky DPCs in cells are proteolytically processed to shorter DNA-peptide cross-links before they can be tolerated by translation synthesis mechanism or removed by nucleotide excision repair. DPCs can block DNA replication, signaling for recruitment of specialized metalloprotease (Spartan). However, the mechanisms of protease-mediated DPC digestion in the absence of DNA replication are incompletely understood. In Chapter VI, we employed an immunoprecipitation(IP)-PCR methodology to demonstrate that DPCs present on non-replicating plasmids are rapidly ubiquitinylated in mammalian cells, which likely serves as a signal for the proteasome- mediated DPC processing or other ubiquitin-mediated pathways to facilitate the DPC repair.
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    DNA-protein cross-linking by bifunctional DNA alkylating agents.
    (2010-03) Michaelson-Richie, Erin Denise
    Many common DNA alkylating agents, such as environmental toxins and chemotherapeutic drugs, are bis-electrophiles capable of covalently cross-linking cellular biomolecules. While DNA-DNA cross-linking by such compounds is well-characterized, the identities and the biological effects of the corresponding DNA-protein cross-links (DPCs) are poorly understood. Furthermore, because bis-electrophiles produce DNA-DNA cross-links and DNA monoadducts in addition to DPCs, it is difficult to establish the biological outcomes specifically resulting from DPC lesions. The purpose of the present work was to characterize DNA-protein cross-linking by two bis-electrophiles, 1,2,3,4-diepoxybutane (DEB) and bis(2-chloroethyl)methylamine (mechlorethamine), and to evaluate the ability of DPCs to induce cytotoxic and mutagenic effects. Mass spectrometry-based proteomics and immunological detection methods identified 41 proteins participating in DPC formation in the presence of DEB in nuclear protein extracts prepared from human cervical carcinoma (HeLa) cells, and 38 proteins which formed DPCs to the chromosomal DNA of human fibrosarcoma (HT1080) cells treated with mechlorethamine. Relative to their cellular abundance, a disproportionately high number of the proteins involved in DPC formation were nuclear proteins with known nucleic acid-binding capabilities which participate in cellular processes such as transcriptional regulation and DNA repair. HPLC-ESI+-MS/MS analysis of total proteolytic digests of DPCs revealed the chemical structures of the cross-links produced by DEB and mechlorethamine to be 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-N7G-BD) and N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA), respectively. In order to analyze the biological consequences of DPC lesions, we selectively induced DPCs in mammalian cell cultures by electroporating them in the presence of epoxide-containing protein reagents. Significant levels of cell death and mutations were observed, suggesting that DPC lesions contribute to the biological effects of bis-electrophiles.
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    Mass Spectrometry-Based Characterization, Quantitation, And Repair Investigations Of Complex DNA Lesions
    (2018-03) Groehler IV, Arnold
    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.