Synthesis and characterization of oligodeoxyribonucleotides containing a site-specifically incorporated N-carboxymethyl-20-deoxyadenosine or N-carboxymethyl-20-deoxycytidine

Humans are exposed to both endogenous and exogenous N-nitroso compounds (NOCs), and many NOCs can be metabolically activated to generate a highly reactive species, diazoacetate, which is capable of inducing carboxymethylation of nucleobases in DNA. Here we report, for the first time, the chemical syntheses of authentic Ncarboxymethyl-20-deoxyadenosine (N-CMdA) and N-carboxymethyl-20-deoxycytidine (N-CMdC), liquid chromatography–ESI tandem MS confirmation of their formation in calf thymus DNA upon diazoacetate exposure, and the preparation of oligodeoxyribonucleotides containing a sitespecifically incorporated N-CMdA or N-CMdC. Additionally, thermodynamic studies showed that the substitutions of a dA with N-CMdA and dC with N-CMdC in a 12-mer duplex increased Gibbs free energy for duplex formation at 25 C by 5.3 and 6.8 kcal/mol, respectively. Moreover, primer extension assay revealed that N-CMdC was a stronger blockade to Klenow fragment-mediated primer extension than N-CMdA. The polymerase displayed substantial frequency of misincorporation of dAMP opposite N-CMdA and, to a lesser extent, misinsertion of dAMP and dTMP opposite N-CMdC. The formation and the mutagenic potential of N-CMdA and N-CMdC suggest that these lesions may bear important implications in the etiology of NOC-induced tumor development. INTRODUCTION Human genome is frequently attacked by various endogenous and exogenous species, and the resulting damage to DNA has been implicated in a number of pathological conditions including cancer and neurological disorders (1). Emerging data have supported a previous hypothesis that DNA damage induced by N-nitroso compounds (NOCs) in the gastric cavity may constitute the early steps of carcinogenesis in gastric cancer due to the high nitrite content of the gastric juice (2,3). In a recent review, Risch (4) proposed that frequent gastrointestinal exposure to NOCs contributes to increased risk of developing pancreatic cancer. In this respect, NOCs induce DNA adducts and single-strand breaks (5), which appear to stimulate DNA synthesis in the pancreatic ductal epithelium (6). Thus, exposure to NOCs may work synergistically with other factors to induce tumor development (7). Humans are exposed to NOCs from diet, tobacco smoke and other environmental sources as well as from endogenous sources, and it was estimated that endogenous sources contribute to 45–75% of the total NOC exposure (3). Earlier studies by Wogan and colleagues (8) revealed that the treatment of male Fischer rats with nitrosated bile acid conjugates N-nitrosoglycocholic acid (NOGC) and N-nitrosotaurocholic acid (NOTC) could induce hepatocarcinoma in 54–70% of the treated animals and gastric tumors in 12–13% of the treated rats. Forward mutation assays showed that NOGC and NOTC are mutagenic in bacteria cells and human lymphoblasts. In this regard, NOGC could arise from the nitrosation of glycocholic acid under simulated gastric conditions (9,10). The treatment of calf thymus DNA with NOGC could induce the formation of O-carboxymethyl-20-deoxyguanosine (O-CMdG) and, *To whom correspondence should be addressed. Tel: 951 827 2700; Fax: 951 827 4713; Email: [email protected] 6774–6784 Nucleic Acids Research, 2010, Vol. 38, No. 19 Published online 27 May 2010 doi:10.1093/nar/gkq458 The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. much less frequently, O-methyl-20-deoxyguanosine (O-MdG) (11). By using immunoslot blot assay or immunoaffinity/HPLC method with antibody recognizing O-carboxymethylguanine, Shuker and coworkers (12–14) showed that O-CMdG could be induced in DNA by N-nitrosoglycine. In addition, O-diazoacetyl-L-serine (also known as azaserine), a pancreatic carcinogen (15), could result in the carboxymethylation of guanine residues in DNA of pancreatic acinar cells (16). The NOC-induced DNA carboxymethylation was thought to occur through a common reactive intermediate, diazoacetate, which was shown to induce the formation of O-CMdG and, much less efficiently (about 1/16 of that of O-CMdG), O-MdG in DNA (12–14). In addition, the passage of potassium diazoacetate (KDA)treated, human p53 gene-containing plasmid in yeast cells could lead to substantial single-base substitutions. Importantly, the mutation spectra of KDA obtained from the shuttle vector study exhibit remarkable similarity as the mutation spectra of p53 gene in human stomach and colorectal cancers (17). This finding underscores that gastrointestinal exposure to NOCs and the resulting generation of diazoacetate may constitute a significant etiological factor for the development of gastrointestinal tumors (17). In addition, KDA induced almost equal frequencies of transition and transversion mutations, which is distinctive from the predominant GC!AT transition mutation (>80%) induced by methylating agent methylnitrosourea (17). Moreover, the percentages of mutations occurring at AT base pairs contribute to 43 and 28% of all observed mutations, while the p53 genecontaining vector was treated with KDA in PBS and Tris–EDTA, respectively. The above observation supports that KDA can induce promutagenic lesions at both GC and AT base pairs in DNA. As discussed above, KDA was found to induce the formation of O-CMdG and we recently reported the formation of N3and O-carboxymethylthymidine in calf thymus DNA upon exposure to KDA (18). However, it remains unexplored what type of DNA lesions can be induced at adenine and cytosine sites in DNA. In the present study, we set out to examine the chemistry of diazoacetate-induced modifications of adenine and cytosine in DNA, and to synthesize oligodeoxyribonucleotides (ODNs) containing carboxymethylated derivatives of 20-deoxyadenosine and 20-deoxycytidine at defined sites. In addition, we will have a preliminary assessment about how these lesions destabilize duplex DNA and how they perturb DNA replication in vitro. MATERIALS AND METHODS