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 mob ility shift, super gel mobility shift, and Western blot to determine the fa te of RPA during Hoechst 33342-induced apoptosis in HL-60 cells. Multiple b ands were detected by gel mobility shift after the incubation of single-str anded gamma-P-32-labeled oligo(dT)(30) with the nuclear extracts of HL-60 c ells. Super gel mobility shift results indicated that only the highest mole cular weight protein/oligo(dT)(30) complexes bound with antihuman RPA-32 an d/or anti-human RPA-70 antibodies forming RPA/oligo(dT)(30) complexes. Afte r the treatment of HL-60 cells with 15 mug/ml Hoechst 33342 for 3 h, the ba nds of RPA/oligo(dT)(30) complexes were decreased and bands of the lowest m olecular weight protein/oligo(dT)(30) complexes were significantly increase d 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-dependen t manner after 1 h of incubation with Hoechst 33342. These results demonstr ate 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 im munoactive RPA-70 and RPA-32. (C) 2001 Academic Press.