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

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 dir ect detection of MDS in DNA. In the present study we demonstrate the potent ial of protocols incorporating an exonucleolytic snake venom phosphodiester ase (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 s ingle 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 spectr ometry. The unambiguous observation of SVPD-resistant 'trimer' species in t he digests of oligonucleotides containing adjacent F, T-g and Me-PTE demons trates that the SVPD digestion strategy is capable of allowing direct detec tion of certain tandem damage. Furthermore, in studies to determine the spe cificity of SVPD in dealing with pairs of lesions on the same strand, it wa s found mandatory to have the two lesions immediately adjacent to each othe r 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.