Synthesis and enzymatic processing of oligodeoxynucleotides containing tandem base damage

Several studies have shown that ionizing radiation generates a wide spectru m of lesions to DNA including base modifications, abasic sites, strand brea ks, crosslinks and tandem base damage. One example of tandem base damage in duced by OH radical in X-irradiated DNA oligomers is N-(2-deoxy-beta-D-eryt hropentofuranosyl)-formylamine/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG uo), In order to investigate the biological significance of such a tandem l esion, both 8-oxo-7,8-dihydroguanine and formylamine were introduced into s ynthetic oligonucleotides at vicinal positions using the solid phase phosph oramidite method. For this purpose, a new convenient method of synthesis of 8-oxodGuo was developed. The purity and integrity of the modified syntheti c DNA fragments were assessed using different complementary techniques incl uding HPLC, polyacrylamide gel electrophoresis, electrospray and MALDI-TOF mass spectrometry. The piperidine test applied to the double modified base- containing oligonucleotides revealed the high alkaline lability of formylam ine in DNA, In addition, various enzymatic experiments aimed at determining biochemical features of such multiply damaged sites were carried out using the synthetic substrates, The processing of the vicinal lesions by nucleas e Fl, snake venom phosphodiesterase, calf spleen phosphodiesterase and repa ir enzymes including Escherichia coli endonuclease (endo) III and Fapy-glyc osylase was studied and is reported.