MEDICAL AND ENVIRONMENTAL EFFECTS OF UV RADIATION - Environmental UV Radiation: Impact on Ecosystems and Human Health and Predictive Models - page 42

several orders of magnitude, very small quantities of DNA are required for a
measurement, and lesions can be measured at very low frequencies. For a
homogeneous DNA, i.e., a molecular species of one size and conformation, less than
one ng DNA can be used. For damage determination in higher organisms, which
contain heterodisperse DNAs, about 50 ng of DNA are used for routine assays [20, 21].
The sensitivity of number average length determination depends on two factors: the size
of the DNA molecules and the minimum fraction of molecules that must be broken to
be detected in the specific combination of biochemical, electrophoretic, imaging system
and computer analysis that is used. For double-strand DNA populations in the size
range of ~1 Mbp, damages can be measured down to ~5/Gpb [22].
A wide variety of damages can be quantitated by this approach. Lesions
affecting one DNA strand are measured by treatment with a lesion-specific cleaving
agent and dispersion under conditions that separate the DNA strands ("denaturing''
conditions), and the resulting frequencies are computed as lesions/base. Damages
affecting both strands are measured by treatment with the agent as above, but with
dispersion under conditions that do not separate the DNA strands, i.e., "non-denaturing''
conditions. Strand breaks, whether single strand or double strand breaks, are measured
directly, since they in themselves comprise breaks of the phosphodiester backbone.
In addition, lesion-specific or lesion class-specific enzymes that cleave the
phosphodiester backbone at each lesion site can be used to convert each lesion to a
strand break. Examples of DNA lesions and the cleavage agents that recognize them
include: cyclobutyl pyrimidine dimers, T4 endonuclease V [12] or Micrococcus luteus
UV endonuclease [23, 24]; oxidized purines Escherichia coli Fpg protein [25]; oxidized
pyrimidines, Escherichia coli Endonuclease III [26]. Many of these enzymes have
multiple substrates, which must be taken into account. For example, the glycosylase
activity of T4 endonuclease V removes the dimer from DNA, and its lyase activity
attacks the resulting abasic site(s) and cleaves the DNA [27]. By its lyase activity, this
enzyme cleaves DNA at 'regular' abasic sites, i.e., a base-less sugar moiety resulting
from cleavage of the N-glycosyl bond to the base [27]. It also recognizes FapyAdenine
residues induced by ionizing radiation and in low yields by UV [13].
5. Action Spectroscopy: Powerful Approach For Understanding UV Effects
Action spectra relate the effectiveness of specific wavelengths in producing an effect.
Figure 4. Action spectrum for induction of cyclobutyl pyrimidine dimers in T7 DNA in solution.