MEDICAL AND ENVIRONMENTAL EFFECTS OF UV RADIATION - Environmental UV Radiation: Impact on Ecosystems and Human Health and Predictive Models - page 40

Number average length analysis can be used to measure lesions affecting one
DNA strand, in which case the DNA is dispersed according to single strand molecular
length using denaturing conditions. For assessment of lesions affecting one strand, in
which DNAs are dispersed on denaturing gels, it is essential to use fully single-stranded
molecules as molecular length standards to establish DNA dispersion curves. Partially
denatured molecules can form branched structures, whose electrophoretic mobility may
not be a direct function of the DNA molecular length. In the case of lesion
determination on denaturing gels, the lesion frequencies are computed in units of
damage are lesions per base, or per kilobase (kb, 10 3 bases), or per megabase (Mb, 10 6
bases), or per gigabase (Gb, 10 9 bases), etc. For determinations on non-denaturing gels,
the damage frequencies are computed in terms of kilobase pairs (kbp), megabase pairs
(Mbp) or gigabase pairs (Gbp).
This method can also measure damages affecting both DNA strands; in this case,
the double strand molecular length standard DNAs are electrophoresed on the same gel
as the experimental samples. Again, no specific distribution of damages is required.
In this case, the damage frequency is given as damages per base pair, per kilobase
pair, or even per gigabase pair. Figure 3 shows the principles of damage

Figure 3. Induction of lesions in a DNA molecule and treatment with a lesion-specific agent.

measurement by these methods. A DNA molecule is damaged by an agent to produce
two lesions shown as solid circles. The population of molecules is divided into two, and
one portion is treated with an agent that makes a DNA strand break at the site of each
lesion. Some examples of such lesion-specific agents are the T4 Endonuclease V,
which—among UV-induced lesions—specifically recognizes cyclobutyl pyrimidine
dimers [12]. (More recently it has been shown that T4 Endo V also recognizes
FapyAdenines, oxidized bases induced by ionizing radiation and at low frequencies by
UV [13].) The other portion is incubated without the lesion-specific treatment. We note
that the treated population now consists of three molecules, while there is only one
molecule in the untreated population. Simple arithmetic shows that the number of
lesions (L, two) can be calculated by subtracting the number of molecules in the
untreated population (N u , one) from the number in the treated population (N t , three).

L = N t - N u (1)

Generally, however, we want to know the Frequency (F) of damages, i.e., lesions per
base or per thousand bases or per million bases. This is readily computed by dividing
each term by the number of bases (b) in the treated and untreated samples, i.e.