Underestimation of the small residual damage when measuring DNA double-strand breaks (DSB): is the repair of radiation-induced DSB complete?
OBJECTIVE
To overcome the underestimation of the small residual damage when measuring DNA double-strand breaks (DSB) as fraction of activity released (FAR) by pulsed-field gel electrophoresis.


METHODS
The techniques used to assess DNA damage (e.g. pulsed-field gel electrophoresis, neutral elution, comet assay) do not directly measure the number of DSB. The Blöcher model can be used to express data as DSB after irradiation at 4 degrees C by calculating the distribution of all radiation-induced DNA fragments as a function of their size. We have used this model to measure the residual DSB (irradiation at 4 degrees C followed by incubation at 37 degrees C) in untransformed human fibroblasts.


RESULTS
The DSB induction rate after irradiation at 4 degrees C was 39.1+/-2.0 Gy(-1). The DSB repair rate obtained after doses of 10 to 80 Gy followed by repair times of 0 to 24 h was expressed as unrepaired DSB calculated from the Blöcher formula. All the damage appeared to be repaired at 24h when the data were expressed as FAR, whereas 15% of DSB remained unrepaired. The DSB repair rate and the chromosome break repair rate assessed by premature condensation chromosome (PCC) techniques were similar.


CONCLUSIONS
The expression of repair data in terms of FAR dramatically underestimates the amount of unrepaired DNA damage. The Blöcher model that takes into account the size distribution of radiation-induced DNA fragments should therefore be used to avoid this bias. Applied to a normal human fibroblast cell line, this model shows that DSB repair is never complete.
Foray
N
N
CNRS UMR 1599-PR1, Institut Gustave-Roussy, Villejuif, France. nforay@igr.fr
Arlett
C F
CF
Malaise
E P
EP
D002460:Cell Line; D019457:Chromosome Breakage; D002877:Chromosomes, Human / Q000528:radiation effects; D003080:Cold Temperature; D004247:DNA / Q000032:analysis* / Q000502:physiology / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D016521:Electrophoresis, Gel, Pulsed-Field; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D006801:Humans
D004247:DNA