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

Abstract

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 ∼72 S to ∼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 O 6-ethyldeoxyguanosine, we studied the influence of chromatin conformation on the formation of O 6-ethylguanine (O 6-EtGua) in the DNA of chromatin exposed to the carcinogen N-ethyl- N-nitrosourea (EtNU; 1 mg/ml, 37°C, 20 min) in vitro. When the NaCl concentration during incubations with EtNU was varied between 0 and 100 mM, the amount of O 6-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 ≧ 50 mM NaCl . A similar dependence on ionic strength was found for the formation of O 6-EtGua in the DNA of Ch − and in native DNA. The frequency of O 6-EtGua was highest in native DNA, followed by the DNA of Ch −, and lowest in the DNA of Ch +. At each salt concentration, the O 6-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 O 6-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 O 6-EtGua and 3-EtAde. As in the case of O 6-EtGua, the frequencies of 7-EtGua and 3-EtAde were dependent on ionic strength, and decreased in the order: native DNA, Ch − DNA, and Ch + DNA. Compared with native DNA (relative value, 100), the frequencies of O 6-EtGua and 7-EtGua in DNA were reduced to a similar extent in Ch − (rel. values 62.1 and 61.2, respectively) and in Ch + (rel. values for both products, 43.9). The corresponding values for 3-EtAde were slightly lower in both types of chromatin fibers (rel. values 56.7 and 39.5, respectively). Thus, the core histones generally protect DNA from ethylation by EtNU. While nucleophilic sites in the major groove and in the base-pairing region of the DNA double helix are protected to about the same degree, the N-3 position of adenine in the minor groove is slightly less accessible to the ethyldiazonium ion generated from EtNU. In all cases the highest degree of protection is obtained when histone H1 is present in chromatin.