Nuclease-induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide.
A flow cytometric analysis of DNA structural changes induced by cleavage with nucleases was performed on isolated HeLa nuclei by assessing changes in stainability with the DNA-specific fluorochrome propidium iodide (PI). After mild digestion with DNAse I, micrococcal nuclease, or with the single-strand-specific S1 and Neurospora crassa nucleases, fluorescence intensity of nuclei stained with PI increased by about 15-30% above the value of undigested control samples. No significant modifications were observed with the restriction enzymes Eco RI, Alu I, and Not I. The DNAse I-induced increase in fluorescence intensity was also observed with the non-intercalating dye Hoechst 33258, but not with mithramycin. Nuclease-induced fluorescence intensity changes as determined with PI were found to be dependent on the dye concentration. A constant increase (about 20%) was measured at dye/DNA-P ratios greater than 0.11. Below this value (2 micrograms/ml PI), the fluorescence intensity of digested samples was 15-30% lower than that of undigested controls. This behaviour towards intercalating dyes is similar to that of the relaxed (nicked) vs. the supercoiled (intact) form of circular DNA. These results suggest that conformation- but not sequence-specific nucleases induce a relaxation of DNA supercoils.
Prosperi
E
E
Dipartimento di Biologia Animale, Università di Pavia, Italy.
Giangarè
M C
MC
Bottiroli
G
G
D004396:Coloring Agents; D004247:DNA / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D004278:DNA, Superhelical / Q000187:drug effects / Q000648:ultrastructure; D003850:Deoxyribonuclease I / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000494:pharmacology*; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D011419:Propidium / Q000378:metabolism
D004396:Coloring Agents; D004278:DNA, Superhelical; D011419:Propidium; D004247:DNA; D003851:Deoxyribonucleases; D003850:Deoxyribonuclease I