Synthesis of single and tandem DNA damages and mutagenesis studies of gamma-radiation-induced cross-linked guanine-thymine tandem lesion

Date of Completion

January 2007


Chemistry, Biochemistry|Chemistry, Organic




The common environmental pollutant 1-nitropyrene and ionizing radiation are two examples of DNA damaging agents. Both these agents are mutagenic, tumorigenic, and carcinogenic. The reductive activation of the carcinogen 1-nitropyrene forms a C8 2'-deoxyguanosine adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (C8-AP-dG), and two N2 2'-deoxyguanosine adducts, 6-(deoxyguanosin-N2-yl)-1-aminopyrene ( N2-6-AP-dG) and 8-(deoxyguanosin-N 2-yl)-1-aminopyrene (N2-8-AP-dG), respectively. Ionizing radiation, on the other hand, gives rise to a large number of DNA damages, including tandem lesions such as a guanine-thymine, G[8,5-Me]T, and thymine-guanine, T[5-Me,8]G, intrastrand cross-links.^ To study the structural and biological effects of these DNA lesions, we used a total synthesis approach to synthesize the N2-AP-dG adducts and cross-links. The DNA adducts or cross-links were incorporated into defined sites of a dodecamer oligodeoxynucleotide, 5'-GTG CGTGTTTGT-3', which contains the local DNA sequence (5'...CGT...3') of the p53 codon 273. In the case of N2-AP-dG adducts, the modification was introduced in the dG residue of CG*T, whereas the cross-link was introduced in the GTG region (i.e., G^Tand T^G).^ Various conditions of Buchwald-Hartwig palladium-catalyzed amination have been examined to synthesize the two N2 2'-deoxyguanosine adducts. The most convenient synthetic approach involved a coupling between the protected 2'-deoxyguanosine and bromonitropyrenes, which, upon reductive deprotection, provided excellent yield of N 2-6-AP-dG and N2-8-AP-dG. The phosphoramidite derivatives of the N2-8- and N2-6-AP-dG adduct were prepared for solid-phase phosphoramidite oligodeoxynucleotide synthesis.^ 5-(Phenylthiomethyl)-2'-deoxyuridine is a photolabile precursor to the 5-(2'-deoxyuridilyl) methyl radical, which generates cross-link with adjacent guanine residues under anaerobic conditions. We synthesized oligodeoxynucleotides containing the cross-links by introducing 5-(phenylthiomethyl)-2'-deoxyuridine by total synthesis followed by UV-C irradiation of the purified oligodeoxynucleotide. The oligodeoxynucleotides containing the cross-links were characterized by using a series of analytical methods.^ The oligodeoxynucleotides containing the cross-links were incorporated into a plasmid vector, which was replicated in human embryonic kidney (293T/17) cells. Our results showed that both T[5-Me,8]G and G[8,5-Me]T lesions are mutagenic. Although the primary mutations induced by both cross-links were targeted G → T transversions, the T[5-Me,8]G cross-link was much more mutagenic than the G[8,5-Me]T cross-link (∼32% versus ∼18%). The targeted G → T transversions induced by T[5-Me,8]G (∼11%) was approximately 4 fold of that induced by G[8,5-Me]T (∼3%). In addition, in both cases semi-targeted mutations occurred at the 5'-adjacent base, which was mutated to T in significant frequency. To our knowledge, this is the first evidence of mutagenicity of these intra-strand cross-links in a cell. ^