Disruption of replication protein A/single-stranded DNA complexes during apoptosis in HL-60 cells.
Replication protein A (RPA) is a single-stranded DNA-binding protein which plays a role in DNA replication, repair, and recombination. We used gel mobility shift, super gel mobility shift, and Western blot to determine the fate of RPA during Hoechst 33342-induced apoptosis in HL-60 cells. Multiple bands were detected by gel mobility shift after the incubation of single-stranded gamma-(32)P-labeled oligo(dT)(30) with the nuclear extracts of HL-60 cells. Super gel mobility shift results indicated that only the highest molecular weight protein/oligo(dT)(30) complexes bound with anti-human RPA-32 and/or anti-human RPA-70 antibodies forming RPA/oligo(dT)(30) complexes. After the treatment of HL-60 cells with 15 microg/ml Hoechst 33342 for 3 h, the bands of RPA/oligo(dT)(30) complexes were decreased and bands of the lowest molecular weight protein/oligo(dT)(30) complexes were significantly increased when compared to the control group. These low-molecular-weight bands did not bind with RPA-32 or RPA-70 antibodies. Western blotting results showed that both RPA-32 and RPA-70 were decreased significantly in a time-dependent manner after 1 h of incubation with Hoechst 33342. These results demonstrate that in HL-60 cells, Hoechst 33342-induced apoptosis is associated with a rapid loss of the binding capacity of RPA to oligo(dT)(30) as well as immunoactive RPA-70 and RPA-32.
Zhang
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Department of Clinical Pathology, William Beaumont Hospital, 3601 West 13 Mile Road, Royal Oak, MI 48073, USA.
Kiechle
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D017209:Apoptosis / Q000187:drug effects; D001562:Benzimidazoles / Q000494:pharmacology; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D015151:Immunoblotting; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D051716:Replication Protein A; D013997:Time Factors
D001562:Benzimidazoles; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D009687:Nuclear Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; C017807:bisbenzimide ethoxide trihydrochloride