An electrophoretic approach to the assessment of the spatial distribution of DNA double-strand breaks in mammalian cells.
An approach is presented making it possible to investigate whether breaks in fragmented mammalian chromosomal DNA were induced randomly and independently from each other. Genomic DNA isolated from mammalian cells 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 bromide-stained gels. The DNA profiles thus obtained were compared to the 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 transformation. Considering either directly measurable DNA mass profiles in units of migration distances along a gel lane or transformed distributions in 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-strand breaks (DSB). Using restriction enzyme-treated DNA as a paradigm, the disagreement of predicted and observed data shows the applicability 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 induction by densely ionizing radiation. Furthermore, investigations on the spatial distribution of chemically or endogenously produced DSB, as well as residual DSB after repair, may be attempted.
Friedl
A A
AA
GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Strahlenbiologie, Oberschleissheim, Germany.
Kraxenberger
A
A
Eckardt-Schupp
F
F
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis* / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D004262:DNA Restriction Enzymes / Q000494:pharmacology; D004271:DNA, Fungal; D004279:DNA, Viral; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D004996:Ethidium; D005720:Gamma Rays; D006801:Humans; D008433:Mathematics
D004271:DNA, Fungal; D004279:DNA, Viral; D004247:DNA; D004262:DNA Restriction Enzymes; D004996:Ethidium