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A Mg(2+)-dependent endonuclease is responsible for internucleosomal DNA fragmentation in human B lymphoblastic IM9
1Department of Biochemistry, College of Science, and Bioproducts Research Center, Yonsei University, Seoul, Korea.
Abstract:
We have identified a Mg(2+)-dependent endonuclease activity from human B lymphoblastic IM9 cell lysates and nuclei using autodigestion method and DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease assay system. The level of the endonuclease activity in cell lysates was significantly decreased at certain stage by treatment of the cells with cycloheximide. However, the enzyme activity consistently remained for over 12 hours in the isolated nuclei of the apoptotic IM9 cells. The Mg(2+)-dependent endonuclease isolated from the nuclei by native-PAGE elution was able to catalyze the conversion of supercoiled plasmid DNA into linear form. This particular endonuclease activity was not detected in cycloheximide treated-U937 cells. Several lines of experimental evidence suggest that the Mg(2+)-dependent endonuclease localized in the nucleus may be responsible for the DNA fragmentation of apoptotic IM9 cells.
Insights
Researchers found a magnesium-dependent endonuclease in human apoptotic cells. This enzyme, localized in the nucleus, is likely responsible for DNA fragmentation during apoptosis.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Apoptosis, or programmed cell death, involves DNA fragmentation.
- The specific enzymes responsible for DNA fragmentation during apoptosis are not fully characterized.
- Understanding these nucleases is crucial for comprehending cell death pathways.
Purpose of the Study:
- To identify and characterize a Mg(2+)-dependent endonuclease involved in apoptosis.
- To investigate the cellular localization and activity of this enzyme.
- To determine its role in DNA fragmentation in human B lymphoblastic IM9 cells.
Main Methods:
- Utilized autodigestion and DNA-native-polyacrylamide gel electrophoresis (DNA-native-PAGE) nuclease assay.
- Assayed endonuclease activity in cell lysates and isolated nuclei.
- Investigated enzyme activity in cycloheximide-treated cells and apoptotic cells.
- Isolated the endonuclease via native-PAGE elution.
Main Results:
- Identified a Mg(2+)-dependent endonuclease activity in IM9 cell lysates and nuclei.
- Enzyme activity decreased in cell lysates upon cycloheximide treatment but persisted in isolated nuclei of apoptotic cells.
- The isolated endonuclease converted supercoiled plasmid DNA to linear form.
- This activity was absent in cycloheximide-treated U937 cells.
Conclusions:
- A Mg(2+)-dependent endonuclease localized in the nucleus of IM9 cells was identified.
- This enzyme is likely responsible for the DNA fragmentation observed during apoptosis in these cells.
- Further research into this endonuclease could reveal new insights into apoptotic mechanisms.