Research Program in Mutagenesis and Carcinogenesis
DNA damage to the nucleoside bases can impact cellular metabolism by inducing copying errors during replication and by increasing genomic instability. These events play a critical role in driving the initiation, progression and metastasis of human cancers. Our laboratory is interested in understanding how specific DNA base damage accumulates under physiological conditions and how it promotes mutagenesis and cancer. These studies typically involve measuring DNA damage levels in vivo, determining the mutagenic profile of specific damage products, and examining the ability of cells to repair them. This effort is highly multidisciplinary, and involves the application of synthetic DNA chemistry, analytical methodologies such as LC-ESI-MS/MS; protein purification; enzyme kinetics, molecular biology, and mammalian tissue culture techniques. Our program offers an excellent training environment for chemistry students seeking to acquire additional skills required for participating in translational and biomedical research.
More than one hundred DNA damage products have been described to date, variously resulting from environmental exposure to radiation or chemicals, or endogenous cellular processes such as oxidative stress, lipid peroxidation and enhanced glycolytic flux. Distinguishing biologically relevant damage from mechanistically interesting chemical artifacts is a constant challenge. We attempt to limit our focus to those DNA damage products which have been detected in mammalian sources and can preferably be correlated with some pathological condition. Some lesions we have examined in the past or are currently studying are shown below.
Modified nucleosides (1) and (3) are products of the reaction of DNA with peroxyl radicals, relatively long-lived reactive intermediates formed during lipid peroxidation. An increase in levels of cellular lipid peroxidation is believed to play an important role in carcinogenesis, mediated in part by free radical reactions of peroxyl radicals with DNA. We showed that peroxyl radicals could oxidize thymine primarily at the C5 Me group, producing mutagenic damage products such as 5-formyl uridine (1) and several other related adducts (Martini and Termini 1997). Peroxidation of important membrane fatty acids such as arachidonic acid (20:4, n-6) was shown to catalyze the oxidation of guanines to 8-oxodG (3) and secondary oxidation products, as well as efficiently induce single-strand breaks in DNA (Lim, Wuenschell et al. 2004).
Nitric oxide (NO), released by neutrophils and macrophages as part of the immune response, can catalyze the oxidative deamination of guanine resulting in the formation of xanthine (2) in DNA. We have studied the miscoding properties of (2), and found that it mainly induces G to A transitions, a prominent feature of NO induced mutagenesis (Wuenschell, O'Connor et al. 2003). We also determined that xanthine in DNA is a substrate for several base excision repair enzymes, including the human methyl purine glycosylase.
Enhanced glycolytic flux can have deleterious effects on proteins and membranes as a result of glycation, a series of complex reactions of carbohydrate-derived aldehydes with biological nucleophiles such as amines and thiols. Biopolymer modification by glycation is a major cause of the diverse pathologies associated with metabolic disease. Glycation can also target DNA, giving rise primarily to diastereomers of a single guanine adduct, identified as CEdG (carboxyethyl -2’-deoxyguanosine, 4). Not much information is currently available on the distribution of CEdG in human tissues, its potential to induce mutations and genomic instability, and its suitability as a substrate for repair in normal and metabolically compromised cells. We have begun analyzing CEdG levels in animal models of metabolic disease, and have recently completed the synthesis of DNA containing CEdG in order to address some of these important issues.
A subsidiary but important goal of these studies is to determine the suitability of these and other biologically relevant DNA damage products as chemical biomarkers of potential utility in disease diagnostics and treatment monitoring.
Factors influencing retroviral mutagenesis
The hypermutable replication of retroviral RNA genomes is well known and is responsible for the extraordinary adaptability and resistance of retroviruses such as HIV to chemotherapeutic challenge. Although the retroviral polymerase error rate and replication frequency play significant roles in generating retroviral genomic diversity, other factors such as RNA base damage and sequence context influence the frequency and distribution of base substitution mutations observed during retroviral replication. For example, the pro-oxidant cellular environment that accompanies viral infection increases the possibility that oxidative damage to RNA contributes to base substitution mutations during replication, particularly in the absence of known cellular mechanisms for the repair of retroviral RNA. We are exploring this hypothesis through a combination of biochemical studies on the coding properties of oxidized bases during copying by reverse transcriptase (RT) and cellular assays designed to reveal viral mutation patterns produced in response to a specific oxidative challenge. We have shown that oxidative base damage, either in the template strand or incoming nucleotide triphosphate, can induce mutations at high frequencies during HIV-1 RT replication (Valentine & Termini 2001). The mutation patterns observed are similar to those found in hypermutable HIV genes such as the envelope glycoprotein. In a model system using MLV infection of HT1080 target cells, large increases in mutation frequencies following a single cycle of replication have been observed following viral challenge with either peroxyl radicals or nitric oxide, and the observed patterns of base substitution mutations have provided direct evidence of RNA base damage. We have recently shown that nucleotide sequence context can influence HIV-1 RT mispairing frequency by ~ 10 fold, primarily by modulating RT binding and dissociation from template/primer duplex (Yamanka & Termini, 2007).