Browsing by Subject "Translesion synthesis"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    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.
  • Thumbnail Image
    Item
    DNA-Protein Cross-links: Formation, Repair, and Inhibition of DNA replication
    (2020-12) Thomforde, Jenna
    DNA-protein crosslinks (DPCs) are ubiquitous DNA lesions that form when cellular proteins become trapped on DNA following exposure to UV light, free radicals, aldehydes, and transition metals. These ultra-bulky lesions are known to disrupt regular DNA cellular machinery, such as replication, transcription, and repair, leading to mutagenesis and carcinogenesis. DPCs can also form endogenously when naturally occurring epigenetic marks (5-formyl cytosine, 5fC) in DNA react with lysine and arginine residues of histones H2A, H3, and H4 to form Schiff base conjugates. However, the understanding of cellular effects on DPCs is not fully understood. The main objective of this thesis was to investigate the effects of DPCs on replication, as well as elucidate mechanisms of repair.In Chapter II, we investigated the local DNA sequence effects on TLS polymerase bypass of 5fC-mediated DNA-peptide crosslinks. Our previous studies revealed that full size DPCs inhibit DNA replication and transcription but can undergo proteolytic cleavage to produce smaller DNA-peptide conjugates. We have shown that when placed in 5'-CXA-3' sequence context (X=5fC-peptide lesion), DNA-peptide crosslinks can be bypassed by human translesion synthesis (TLS) polymerases ƞ and k in an error-prone manner. However, local nucleotide sequence context can have a large effect on replication bypass of bulky lesions by influencing the geometry of the ternary complex between DNA template, polymerase, and the incoming dNTP. In this chapter, model hydrolytically stable DpCs were prepared by oxime ligation between 5fC in DNA and oxy-lysine containing peptides. Primer extension products were analyzed by gel electrophoresis, and steady state kinetics of dAMP incorporation opposite the DpC lesion in different base sequence contexts was investigated. Our results revealed a strong impact of nearest neighbor base identity on polymerase ƞ activity both in the absence and presence of a DpC lesion. Molecular modeling and molecular dynamics simulations of the hPol η ternary complex, containing the DNA template-primer strands with incoming dATP opposite DpC or unmodified C explained structurally how the nature of the 5' and 3' neighbors of this template profoundly impacted its alignment in the C-A mismatch. Our results reveal an important role of base sequence context in promoting TLS related mutational hotspots both in the presence and in the absence of DpC lesions. In Chapter III, we investigated the role of replicative DNA polymerases δ and ε in DpC lesion bypass. TLS polymerase switches are known to be the primary mechanism to bypass bulky DNA lesions such as DNA-peptide crosslinks, however, DpC-containing plasmids were still replicated at relatively high efficiency in TLS-deficient cell lines, leading to the hypothesis that replicative polymerases are also involved in lesion bypass, in a minor role. In Chapter IV, we employed a sensitive nanoLC-ESI+-LC-MS/MS assay to investigate the formation of ROS-induced DPCs between thymidine in DNA and tyrosine in proteins. This methodology was used to analyze the role of metalloprotease Spartan in repair of ROS-induced DPCs in cells and mouse tissues. A 1.5-2 fold increase of thymidine-tyrosine adducts were detected in the brain, heart, livers, and kidneys of Spartan hypomorphic (SPRTNf/-) mice compared to wild type (SPRTN+/+), providing evidence that Spartan plays a direct role in the repair of ROS-induced DPCs.
  • Thumbnail Image
    Item
    Mass spectrometry-based analysis of urinary metabolites of 1,3-Butadiene (BD) in humans and influence of BD-DNA adducts on DNA replication
    (2013-10) Kotapati, Srikanth
    Cigarette smoking is a known risk factor for the development of lung cancer: approximately 1 out of 5 heavy smokers will develop the disease. However, there are significant differences in risk of lung cancer among smokers from different ethnic/racial groups. African American and Native Hawaiian smokers are at a higher risk of lung cancer than European American, Japanese American or Latin American smokers. Cigarette smoke has more than 70 known carcinogens. Following metabolic activation to electrophilic species, these carcinogens can form covalent DNA adducts, which are capable of inducing heritable mutations ultimately resulting in lung cancer. It has been hypothesized that the observed ethnic/racial differences in lung cancer risk in smokers are due to different frequencies of specific polymorphisms in drug metabolizing genes, leading to a different degree of carcinogen bioactivation to DNA-reactive intermediates. 1,3-Butadiene (BD) is among the most abundant and potent carcinogens present in cigarette smoke. BD is metabolically activated primarily by CYP2E1 to form 3,4-epoxy-1-butene (EB), hydroxymethyl vinylketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB). EB, HMVK, EBD, and DEB have been shown to modify DNA bases to form promutagenic adducts. Alternatively, EB, EBD, and DEB can undergo detoxification via epoxide hydrolysis (the main pathway in humans) or glutathione conjugation and further metabolic conversion into urinary mercapturic acids, 1-hydroxy 2-(N-acetylcysteinyl)-3-butene (MHBMA), 1,2-dihydroxy-4-(N-acetyl cysteinyl)-butane (DHBMA), 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA), and 1,4-bis-(N-acetylcysteinyl)butane-2,3-diol (bis-BDMA), respectively. The research presented in this thesis focuses on revealing any ethnic/racial differences in metabolism of BD in smokers and examining the ability of BD-DNA adducts to cause mutations. In the first part of the thesis, we have identified two novel metabolites of BD which have not been previously detected in vivo: 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA), and 1,4-bis-(N-acetylcysteinyl)butane-2,3-diol (bis-BDMA). To enable their detection in smokers, sensitive and specific HPLC-ESI--MS/MS methods were developed for both metabolites in human urine. We observed significant amounts of THBMA in samples from smokers, non-smokers and occupationally exposed workers. In contrast, bis-BDMA amounts in urine of smokers and occupationally exposed workers were below the method's limit of detection, although it was found in urine of F344 rats exposed to 62.5 or 200 ppm BD. Additionally we found significant interspecies differences in BD metabolism between laboratory rats and humans. DHBMA accounted for only 47% of BD urinary mercapturic acids in rats while the corresponding percentage in humans is 93%. We further developed a high throughput HPLC-ESI--MS/MS method for the quantification of MHBMA and DHBMA in humans and applied this method to quantify urinary BD-mercapturic acids metabolites in workers from a BD and styrene butadiene rubber (SBR) manufacturing facility and smokers belonging to different ethnic groups in two separate multi-ethnic cohort studies. Workers occupationally exposed to BD excreted significantly more BD-mercapturic acids than administrative workers at the same plant. In a small multi-ethnic study of smokers belonging to European American, Native Hawaiian and Japanese American (N = 200 per group), mean urinary MHBMA and MHBMA/DHBMA+MHBMA metabolic ratio were highest in European American and lowest in Japanese American smokers. Similar results were obtained in the larger study (N = 450 per group) composed of European American and African American smokers. Urine of European American smokers contained higher concentrations of MHBMA than that of African Americans. Genome-wide association study (GWAS) analysis conducted for the larger multi-ethnic group has revealed significant associations between single-nucleotide polymorphisms (SNPs) in chromosome 22 (22564172bp - 22735492 bp, nearby genes GSTT1, GSTT2, DDT and MIF) and urinary BD-mercapturic acid levels in smokers, providing the first evidence for genetic and ethnic/racial differences in metabolism of BD.The second part of my thesis work has focused on evaluating the mutagenic ability of three recently discovered BD-dA lesions: N6-(2-hydroxy-3-buten-1-yl)-adenine (N6-HB-dA), N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N6,N6-DHB-dA), and 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-&gamma-HMHP-dA). In vitro translesion synthesis experiments were performed on synthetic oligonucleotides containing each of the three lesions at a site-specific position by gel electrophoresis and HPLC-MS/MS. We found that human translesion synthesis (TLS) polymerases hPols &eta, &kappa, &iota and human polymerase &beta were able to bypass (S)-N6-HB-dA in an error-free manner because of the conserved Watson-Crick base pairing with dT. However, replication past both (R,R)-N6,N6-DHB-dA and (R,S)-1,N6-&gamma-HMHP-dA lesions by TLS polymerases hPols &eta and &kappa was highly error-prone, resulting in A&rarrT, A&rarrC mutations and frameshift deletions. This is the first study that identifies (R,R)-N6,N6-DHB-dA and (R,S)-1,N6-&gamma-HMHP-dA as BD-DNA adducts potentially responsible for the induction of A*rarrT mutations by BD.