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© The Rockefeller University Press, 0021-9525/2000//799 $5.00
The Journal of Cell Biology, Volume 149, Number 4, , 2000 799-810

Original Article

Formation of a Complex between Nucleolin and Replication Protein a after Cell Stress Prevents Initiation of DNA Replication



Yaron Danielya and James A. Borowieca

a Department of Biochemistry, and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016
Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, NY 10016.(212) 263-8166(212) 263-8453

james.borowiec{at}med.nyu.edu

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.

Key Words: replication protein A • nucleolin • nucleolus • SV-40 • heat shock

© 2000 The Rockefeller University Press

Abbreviations used in this paper: AMD, actinomycin D; BrdU, bromodeoxyuridine; EcoSSB, E. coli SSB; GST, glutathione S-transferase; hRPA, human replication protein A; ori, SV-40 origin of replication; pre-rRNA, precursor ribosomal RNA; RIP, RPA-interacting protein; RPA, replication protein A; SSB, single-stranded DNA-binding protein; ssDNA, single-stranded DNA; T antigen, SV-40 large tumor antigen.


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