Aa. Friedl et al., AN ELECTROPHORETIC APPROACH TO THE ASSESSMENT OF THE SPATIAL-DISTRIBUTION OF DNA DOUBLE-STRAND BREAKS IN MAMMALIAN-CELLS, Electrophoresis, 16(10), 1995, pp. 1865-1874
An approach is presented making it possible to investigate whether bre
aks in fragmented mammalian chromosomal DNA were induced randomly and
independently from each other. Genomic DNA isolated from mammalian cel
ls irradiated with gamma-rays or restriction enzyme-treated human DNA
was resolved according to size using pulsed field gel electrophoresis,
and the resulting DNA mass distributions were measured in ethidium br
omide-stained gels. The DNA profiles thus obtained were compared to th
e predictions on DNA fragment size distribution which follow from a so
-called random breakage model to test whether the experimental outcome
is compatible with the assumption of a random localization of breaks.
Comparisons of fragment distributions may be performed utilizing two
equivalent representations that are linked by an adequate transformati
on. Considering either directly measurable DNA mass profiles in units
of migration distances along a gel lane or transformed distributions i
n units of molecular length, we show for gamma-irradiated samples that
the predictions derived from the employed models agree well with the
observed data, thus allowing an immediate quantification of double-str
and breaks (DSB). Using restriction enzyme-treated DNA as a paradigm,
the disagreement of predicted and observed data shows the applicabilit
y of our approach to the detection of a non-random distribution of DSB
. Therefore, we suppose that our approach may also be useful to reveal
a clustering of DSB, which is postulated to occur after damage induct
ion by densely ionizing radiation. Furthermore, investigations on the
spatial distribution of chemically or endogenously produced DSB, as we
ll as residual DSB after repair, may be attempted.