DNA DOUBLE-STRAND BREAKS IN MAMMALIAN-CELLS EXPOSED TO GAMMA-RAYS ANDVERY HEAVY-IONS - FRAGMENT-SIZE DISTRIBUTIONS DETERMINED BY PULSED-FIELD GEL-ELECTROPHORESIS

The spatial distribution of DNA double-strand breaks (DSB) was assesse d after treatment of mammalian cells (V79) with densely ionizing radia tion. Cells were exposed to beams of heavy charged particles (calcium ions: 6.9 MeV/u, 2.1 . 10(3) keV/mu m; uranium ions: 9.0 MeV/u, 1.4 10 (4) keV/mu m) at the linear accelerator UNILAC of GSI, Darmstadt. DNA was isolated in agarose plugs and subjected to pulsed-field gel electr ophoresis under conditions that separated DNA fragments of size 50 kbp to 5 Mbp. The measured fragment distributions were compared to those obtained after gamma-irradiation and were analyzed by means of a convo lution and a deconvolution technique. In contrast to the finding for g amma-radiation, the distributions produced by heavy ions do not corres pond to the random breakage model. Their marked overdispersion and the observed excess of short fragments reflect spatial clustering of DSB that extends over large regions of the DNA, up to several mega base pa irs (Mbp). At fluences of 0.75 and 1.5/mu m(2), calcium ions produce n early the same shape of fragment spectrum, merely with a difference in the amount of DNA entering the gel; this suggests that the DNA is fra gmented by individual calcium ions. At a fluence of 0.8/mu m(2) uraniu m ions produce a profile that is shifted to smaller fragment sizes in comparison to the profile obtained at a fluence of 0.4/mu m(2); this s uggests cumulative action of two separate ions in the formation of fra gments. These observations are not consistent with the expectation tha t the uranium ions, with their much larger LET, should be more likely to produce single particle action than the calcium ions. However, a co nsideration of the greater lateral extension of the tracks of the fast er uranium ions explains the observed differences; it suggests that th e DNA is closely coiled so that even DNA locations several Mbp apart a re usually not separated by less than 0.1 or 0.2 mu m.