Influence of chromatin structure on the induction of DNA double strand breaks by ionizing radiation

Abstract

Pulsed field gel electrophoresis was used to examine the influence of chromatin structure on the induction of DNA double strand breaks by gamma-irradiation in CHO-WBL cells, nuclei, and a series of protein-depleted chromatin substrates. We developed a method to isolate intact nuclei in agarose plugs that avoids DNA shearing and nucleolytic degradation during sample preparation, and facilitates nuclear protein extraction. Agarose plug-isolated nuclei are extracted with increasing concentrations of NaCl to selectively strip off: (a) nonhistone chromosomal proteins (NHP); (b) NHP and histone H1; (c) NHP, H1, and histone H2A-H2B dimers; or (d) NHP, H1, and H2A-H2B dimers and histone H3-H4 tetramers. Following treatment with up to 40 Gy of gamma-radiation, DNA from each sample is purified and the relative induction of DNA double strand breaks is assayed by asymmetric field inversion gel electrophoresis. At a dose of 20 Gy, removal of nonhistone proteins from nuclei results in a 3-fold increase in DNA double strand breaks, compared to intact CHO cells. Additional stripping of histone H1 results in an incremental increase in double strand break induction, whereas further removal of H2A-H2B dimers yields a greater than 10-fold increase in DNA double strand breaks compared to intact CHO cells. The dose-response profile for this latter sample is similar to that observed for purified DNA. These data indicate that distinct classes of chromosomal proteins afford the DNA with different levels of protection against gamma-ray-induced DNA double strand breaks. Thus, chromatin domains that differ in tertiary structure and protein composition may also differ in their susceptibility to DNA double strand breaks induced by ionizing radiation and, perhaps, other clastogens.