Ethylation of nucleophilic sites in DNA by N-ethyl-N-nitrosourea depends on chromatin structure and ionic strength

Depending on ionic strength, chromatin can assume either a condensed, supranucleosomal conformation or the form of an extended nucleosomal fiber. Using sedimentation velocity analysis, both types of structures could be identified in chromatin prepared from cell nuclei of fetal rat brain. When the ionic strength was reduced from 60 to 10 mM NaCl, the average S-value of a defined chromatin fiber fraction (12-15 nucleosomes in size) decreased dramatically from approximately 72 S to approximately 55 S, reflecting the unfolding of condensed chromatin to an extended conformation. Correspondingly, the average S-value of histone H1-depleted chromatin (Ch-) was 54 S at 60 mM NaCl and did not change significantly at lower NaCl concentrations. Ch- contains only the core histones and is, therefore, relaxed into an extended form. Using a monoclonal antibody (ER-6) specific for O6-ethyldeoxyguanosine, we studied the influence of chromatin conformation on the formation of O6-ethylguanine (O6-EtGua) in the DNA of chromatin exposed to the carcinogen N-ethyl-N-nitrosourea (EtNU; 1 mg/ml, 37 degrees C, 20 min) in vitro. When the NaCl concentration during incubations with EtNU was varied between 0 and 100 mM, the amount of O6-EtGua formed in the DNA of complete chromatin (Ch+) was highest at 0 mM NaCl, then decreased exponentially with increasing ionic strength, and remained approximately constant at values greater than or equal to 50 mM NaCl. A similar dependence on ionic strength was found for the formation of O6-EtGua in the DNA of Ch-, and in native DNA. The frequency of O6-EtGua was highest in native DNA, followed by the DNA of Ch-, and lowest in the DNA of Ch+. At each salt concentration, the O6-EtGua content of Ch+ DNA relative to the corresponding values for Ch- DNA and native DNA, remained unchanged (0.70 +/- 0.03 S.D. and 0.42 +/- 0.03 S.D., respectively). In addition to O6-EtGua, the formation of 7-ethylguanine (7-EtGua; major groove of the DNA double helix) and 3-ethyladenine (3-EtAde; minor groove) was analysed after exposure to [1-14C]EtNU. 7-EtGua was the most frequently formed ethylation product, followed by O6-EtGua and 3-EtAde.(ABSTRACT TRUNCATED AT 400 WORDS)