Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1.
Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.
Prosperi
E
E
Centro di Studio per l'Istochimica del C.N.R., Dipartimento di Biologia Animale, Universita' di Pavia, Italy.
Giangaré
M C
MC
Supino
R
R
Bottiroli
G
G
D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D006868:Hydrolysis; D008722:Methods; D011687:Purines / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D002843:Chromatin; D005456:Fluorescent Dyes; D011687:Purines; D012965:Sodium Chloride; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases