Formation of a complex between nucleolin and replication protein A after cell stress prevents initiation of DNA replication.
We used a biochemical screen to identify nucleolin, a key factor in ribosome biogenesis, as a high-affinity binding partner for the heterotrimeric human replication protein A (hRPA). Binding studies in vitro demonstrated that the two proteins physically interact, with nucleolin using an unusual contact with the small hRPA subunit. Nucleolin significantly inhibited both simian virus 40 (SV-40) origin unwinding and SV-40 DNA replication in vitro, likely by nucleolin preventing hRPA from productive interaction with the SV-40 initiation complex. In vivo, use of epifluorescence and confocal microscopy showed that heat shock caused a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm. Nucleolin relocalization was concomitant with a tenfold increase in nucleolin-hRPA complex formation. The relocalized nucleolin significantly overlapped with the position of hRPA, but only poorly with sites of ongoing DNA synthesis. We suggest that the induced nucleolin-hRPA interaction signifies a novel mechanism that represses chromosomal replication after cell stress.
Daniely
Y
Y
Department of Biochemistry, and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, NY 10016, USA.
Borowiec
J A
JA
D002451:Cell Compartmentation; D002466:Cell Nucleolus; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018869:Heat-Shock Response; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D015530:Nuclear Matrix; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018741:Replication Origin; D051716:Replication Protein A; D014407:Tumor Cells, Cultured; D000097796:Nucleolin
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D051716:Replication Protein A; D000097796:Nucleolin; C497167:RPA1 protein, human