Nucleic Acids Research Advance Access originally published online on November 28, 2008
Nucleic Acids Research 2009 37(2):336-345; doi:10.1093/nar/gkn946
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Nucleic Acids Research, 2009, Vol. 37, No. 2 336-345
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chemistry and Synthetic Biology |
Chemical synthesis of oligodeoxyribonucleotides containing N3- and O4-carboxymethylthymidine and their formation in DNA
Department of Chemistry, University of California, Riverside, CA 92521-0403, USA
*To whom correspondence should be addressed. Tel: +1 951 827 2700; Fax: +1 951 827 4713; Email: yinsheng.wang{at}ucr.edu
Received September 11, 2008. Revised November 4, 2008. Accepted November 8, 2008.
Humans are exposed to N-nitroso compounds from both endogenous and exogenous sources. Many N-nitroso compounds can be metabolically activated to give diazoacetate, which can result in the carboxymethylation of DNA. The remarkable similarity in p53 mutations found in human gastrointestinal tumors and in shuttle vector studies, where the human p53 gene-containing vector was treated with diazoacetate and propagated in yeast cells, suggests that diazoacetate might be an important etiological agent for human gastrointestinal tumors. The O6-carboxymethyl-2'-deoxyguanosine was previously detected in isolated DNA upon exposure to diazoacetate and in blood samples of healthy human subjects. The corresponding modifications of thymidine and 2'-deoxyadenosine have not been assessed, though significant mutations at A:T base pairs were found in the p53 tumor suppressor gene in human gastrointestinal tumors and in shuttle vector studies. To understand the implications of the carboxymethylation chemistry of thymidine in the observed mutations at A:T base pairs, here we synthesized authentic N3-carboxymethylthymidine (N3-CMdT) and O4-carboxymethylthymidine (O4-CMdT), incorporated them into DNA, and demonstrated, for the first time, that they were the major carboxymethylated derivatives of thymidine formed in calf thymus DNA upon exposure to diazoacetate. The demonstration of the formation of N3-CMdT and O4-CMdT in isolated DNA upon treatment with diazoacetate, together with the preparation of authentic oligodeoxyribonucleotide substrates housing these two lesions, laid the foundation for investigating the replication and repair of these lesions and for understanding their implications in the mutations observed in human gastrointestinal tumors.