Regulation of dna replication after heat shock by replication protein a-nucleolin interactions.
Heat shock inhibits replicative DNA synthesis, but the underlying mechanism remains unknown. We investigated mechanistic aspects of this regulation in melanoma cells using a simian virus 40 (SV40)-based in vitro DNA replication assay. Heat shock (44 degrees C) caused a monotonic inhibition of cellular DNA replication following exposures for 5-90 min. SV40 DNA replication activity in extracts of similarly heated cells also decreased after 5-30 min of exposure, but returned to near control levels after 60-90 min of exposure. This transient inhibition of SV40 DNA replication was eliminated by recombinant replication protein A (rRPA), suggesting a regulatory process targeting this key DNA replication factor. SV40 DNA replication inhibition was associated with a transient increase in the interaction between nucleolin and RPA that peaked at 20-30 min. Because binding to nucleolin compromises the ability of RPA to support SV40 DNA replication, we suggest that the observed interaction reflects a mechanism whereby DNA replication is regulated after heat shock. The relevance of this interaction to the regulation of cellular DNA replication is indicated by the transient translocation in heated cells of nucleolin from the nucleolus into the nucleoplasm with kinetics very similar to those of SV40 DNA replication inhibition and of RPA-nucleolin interaction. Because the targeting of RPA by nucleolin in heated cells occurs in an environment that preserves the activity of several essential DNA replication factors, active processes may contribute to DNA replication inhibition to a larger degree than presently thought. RPA-nucleolin interactions may reflect an early step in the regulation of DNA replication, as nucleolin relocalized into the nucleolus 1-2 h after heat exposure but cellular DNA replication remained inhibited for up to 8 h. We propose that the nucleolus functions as a heat sensor that uses nucleolin as a signaling molecule to initiate inhibitory responses equivalent to a checkpoint.
Wang
Y
Y
Department of Radiation Oncology, Division of Experimental Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Guan
J
J
Wang
H
H
Wang
Y
Y
Leeper
D
D
Iliakis
G
G
D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018869:Heat-Shock Response; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000473:pathology; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051716:Replication Protein A; D014407:Tumor Cells, Cultured; D000097796:Nucleolin
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D051716:Replication Protein A; D000097796:Nucleolin; C497167:RPA1 protein, human