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09:14
Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique
Published on: January 14, 2016
Disruption of replication protein A/single-stranded DNA complexes during apoptosis in HL-60 cells
1Department of Clinical Pathology, William Beaumont Hospital, 3601 West 13 Mile Road, Royal Oak, MI 48073, USA.
Biochemical and Biophysical Research Communications
|September 28, 2001
Summary
Hoechst 33342 treatment rapidly reduces the DNA-binding ability of Replication Protein A (RPA) and its protein levels during apoptosis in HL-60 cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Replication protein A (RPA) is crucial for DNA replication, repair, and recombination.
- Apoptosis is a programmed cell death process involving significant cellular changes.
Purpose of the Study:
- To investigate the impact of Hoechst 33342-induced apoptosis on RPA function and levels in HL-60 cells.
Main Methods:
- Gel mobility shift assays to detect protein-DNA complexes.
- Super gel mobility shift assays with anti-RPA antibodies.
- Western blotting to quantify RPA protein levels.
Main Results:
- Hoechst 33342 treatment decreased RPA/oligo(dT)(30) complexes and increased non-RPA complexes.
- Both RPA-32 and RPA-70 protein levels significantly decreased over time.
- Loss of RPA binding capacity correlated with apoptosis induction.
Conclusions:
- Hoechst 33342-induced apoptosis in HL-60 cells involves a rapid loss of RPA's DNA-binding capacity.
- The study demonstrates a time-dependent decrease in immunoactive RPA-70 and RPA-32 during apoptosis.
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