DNase I sensitivity of nuclear DNA measured by flow cytometry

Abstract

The DNase I digestion kinetics of DNA in isolated nuclei (from HeLa or murine mammary carcinoma, 67 cells) were assayed flow cytometrically by measuring the changes in ethidium bromide (EtBr) fluorescence following various digestion time intervals. The DNase I digestion curve was characterized by an initial 25–30% increase in fluorescence upon addition of the enzyme, a rapid reduction in fluorescence to approximately 50–55% in 30 minutes, and a limit digest of 45–50% beyond 45 minutes. Throughout digestion, the DNA histogram retained its characteristic bimodal shape, showing that histogram rearrangement was not responsible for the changes in EtBr fluorescence. Irradiation with 5 ± 10⁶ rads (¹³⁷Cs‐γ‐rays) or exposure to 50 mM EDTA caused an increase in EtBr fluorescence similar to that caused by DNase I, suggesting that DNA nicking and/or chromatin loosening were responsible for this increase. Residual DNA assayed by the solubilization of ¹⁴C‐TdR (thymidine)‐labeled DNA indicated a similar kinetic pattern without the initial increase. However, at the limit digest, the fraction of DNA remaining trichloroacetic acid (TCA) insoluble (10%) was smaller than that measured by loss of EtBr fluorescence (50% of initial, 40% of maximum). Part of this difference was due to the presence of TCA soluble DNA trapped within the nuclear matrix (15–20%). This trapped DNA was released when the digested nuclei were exposed to 0.5–1.0 M NaCl just prior to EtBr staining. Exposure of HeLa cells to three agents that are believed to cause changes in chromatin structure resulted in alterations in the DNase I digestion kinetics measured flow cytometrically. N‐butyrate exposure resulted in an increased rate of digestion. In contrast, heat shock and hypertonic shock resulted in a reduced rate of digestion. The proliferating status of the cells was reflected in the DNase I sensitivity of nuclear DNA. Nuclei from quiescent 67 cells showed a 22% reduction in EtBr fluorescence after 10 minutes of digestion, whereas nuclei from proliferating 67 cells showed a 26% loss. Additionally, the initial increase in fluorescence intensity was greater for quiescent cells, possibly reflecting a greater degree of chromatin loosening. Thus, the flow cytometric assay of DNase I sensitivity has been demonstrated to be a useful probe for chromatin structure.