Repair of strand breaks in superhelical DNA of ataxia telangiectasia lymphoblastoid cells.

Abstract

A number of different assay methods have been used to study repair of strand breaks in DNA after exposure of cells to ionizing radiation. Use of these methods indicates that fibroblasts from patients with ataxia telangiectasia (AT), a multiform genetic disease exhibiting high sensitivity to ionizing radiation, have a normal ability to repair strand breaks in DNA. All of these methods determine the extent of breakage of DNA and the resealing of these breaks but do not provide information on restoration of DNA configuration in the nucleus. In this report we have used a sensitive technique to investigate restoration of the 3-dimensional structure of DNA in AT lymphoblastoid cells after exposure to ionizing radiation. This technique provides a means of lysing cells using a high concentration of salt and a non-ionic detergent, giving rise to structures called nucleoids which contain nuclear RNA and DNA, are depleted in protein, and sediment in a manner characteristic of supercoiled DNA. We have shown that the degree of supercoiling is the same in control and AT lymphoblastoid cells using sedimentation in the presence of ethidium bromide. The extent of breakage after exposure of cells to gamma-radiation, and the rate of repair of these breaks are similar in both cell types. Rate of repair of strand breaks is dose dependent and the restructured rapidly sedimenting complex behaves similarly, on sucrose gradients containing ethidium bromide, to that extracted from unirradiated cells.