Evaluation of phosphodiesterase I-based protocols for the detection of multiply damaged sites in DNA: the detection of abasic, oxidative and alkylative tandem damage in DNA oligonucleotides

摘要

It has been proposed that DNA multiply damaged sites (MDS), where more than one moiety in a local region (similar to1 helical turn, 10 bp) of the DNA is damaged, are lesions of enhanced biological significance. However, other than indirect measures, there are few analytical techniques that allow direct detection of MDS in DNA. In the present study we demonstrate the potential of protocols incorporating an exonucleolytic snake venom phosphodiesterase (SVPD) digestion stage to permit the direct detection of certain tandem damage, in which two lesions are immediately adjacent to each other on the same DNA strand. A series of prepared oligonucleotides containing either single or pairs of tetrahydrofuran moieties (F), thymine glycol lesions (T-g) or methylphosphotriester adducts (Me-PTE) were digested with SVPD and the digests examined by either P-32-end-label ling or electrospray mass spectrometry. The unambiguous observation of SVPD-resistant 'trimer' species in the digests of oligonucleotides containing adjacent F, T-g and Me-PTE demonstrates that the SVPD digestion strategy is capable of allowing direct detection of certain tandem damage. Furthermore, in studies to determine the specificity of SVPD in dealing with pairs of lesions on the same strand, it was found mandatory to have the two lesions immediately adjacent to each other in order to generate the trimer species; pairs of lesions separated by as few as one or two normal nucleotides behave principally as single lesions towards SVPD.