Related Experiment Video
Updated: Aug 14, 2026
Use of the Pyrimidine Analog, 5-Iodo-2′-Deoxyuridine (IdU) with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform
Published on: October 22, 2021
Nuclease-induced DNA structural changes assessed by flow cytometry with the intercalating dye propidium iodide
E Prosperi1, M C Giangarè, G Bottiroli
1Dipartimento di Biologia Animale, Università di Pavia, Italy.
Abstract:
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.
Insights
Flow cytometry revealed that conformation-specific nucleases, like DNAse I, alter DNA structure, increasing propidium iodide (PI) dye binding in HeLa nuclei. This suggests DNA relaxation, not sequence cleavage, affects DNA stainability.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nucleases are enzymes that cleave DNA.
- DNA structure and conformation influence its accessibility to dyes.
- Flow cytometry is a technique used to analyze DNA content and structure.
Purpose of the Study:
- To investigate DNA structural changes induced by various nucleases.
- To assess the impact of nuclease activity on DNA stainability with fluorescent dyes.
- To differentiate between conformation-specific and sequence-specific nuclease effects on DNA.
Main Methods:
- Flow cytometry was used to analyze isolated HeLa nuclei.
- Nuclei were digested with different nucleases: DNAse I, micrococcal nuclease, S1 nuclease, Neurospora crassa nuclease, Eco RI, Alu I, and Not I.
- Changes in DNA stainability were assessed using propidium iodide (PI) and Hoechst 33258, with mithramycin as a control.
Main Results:
- Mild digestion with DNAse I, micrococcal nuclease, S1, and N. crassa nucleases increased PI fluorescence intensity by 15-30%.
- Restriction enzymes (Eco RI, Alu I, Not I) did not significantly alter fluorescence.
- DNAse I-induced changes were observed with Hoechst 33258 but not mithramycin, indicating a conformational effect.
- PI fluorescence changes were dye concentration-dependent, mimicking relaxed vs. supercoiled DNA behavior.
Conclusions:
- Conformation-specific nucleases, not sequence-specific ones, induce DNA relaxation.
- DNA structural relaxation increases accessibility to intercalating dyes like PI.
- Flow cytometry can detect nuclease-induced DNA conformational changes.
More Related Videos
06:44Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging
Published on: April 28, 2021
07:09High-throughput Flow-cytometry Measurement of Cellular Mechanotype Based on Rupture and Delivery of DNA Tension Probes into Cells
Published on: June 13, 2025