Abstract: We have developed a new-conceptual analytical methodology to estimate the DNA
damage spectrum on natural DNA without radioisotope and fluorescent labeling.
DNA damage is generally classified into two categories: one is ‘strand break’, the
other is ‘nucleobase lesion’. There are two kinds of termini in the strand break pattern:
the termini with or without phosphate. We have developed the protocols to quantify
3’termini without phosphate (site 1), 3’termini with phosphate (site 2), and nucleobase
lesions (site 3). An enzyme, phosphodiestrase I (snake venom phosphodiesterase
(SVPD)), can recognize a 3’terminus without phosphate followed by production of DNA
monomers (2’-deoxynucleoside-5’-phosphate) sequentially from the 3’terminus (3’ → 5’
exonuclease function). Then, the yield of ‘site 1’ can be quantified since the amount of
the DNA monomers produced during incubation for a given period is proportional to that
of ‘site 1’. In addition, pre-treatment of irradiated DNA by another enzyme, calf intestine
alkaline phosphatase (CIAP) enables ‘site 2’ to be recognized by SVPD, because CIAP
removes phosphate at ‘site 2’ to convert into 3’OH terminus categorized in ‘site 1’.
Furthermore, pre-treatment of irradiated DNA by a chemical, piperidine, can covert most
electron-withdrawing nucleobase lesions into ‘site 2’, which can become recognizable by
SVPD after CIAP pre-treatment as mentioned above. As a result, in the case of 60Co γ-
irradiated dry DNA, the yields of total 3’termini, 3’termini without phosphate, 3’termini
with phosphate, and piperidine-labile nucleobase lesions, are estimated to be 0.102,
0.024, 0.078, and 0.084, respectively. The de novo analytical protocol is unique in the
idea itself, and future analyses based on the methodology will elucidate unknown DNA
damage spectrum using a variety of combinations of enzymes.