Abstract
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.
Highlights
DNA replication is at the center of the eukaryotic cell cycle, and its regulation under conditions of stress, or after exposure to DNA-damaging agents, is under the control of intricate pathways that are only beginning to be characterized
Because binding to nucleolin compromises the ability of replication protein A (RPA) to support simian virus 40 (SV40) DNA replication, we suggest that the observed interaction reflects a mechanism whereby DNA replication is regulated after heat shock
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
Summary
DNA replication is at the center of the eukaryotic cell cycle, and its regulation under conditions of stress, or after exposure to DNA-damaging agents, is under the control of intricate pathways that are only beginning to be characterized. The assay provides a well controlled and molecularly defined system to study regulatory aspects of DNA replication, and allows the separation of regulatory effects targeting DNA replication factors from effects of heat on the substrate DNA. Using this assay we reported [27] that cytoplasmic extracts prepared from heated cells had a reduced DNA replication activity, as compared with extracts prepared from non-heated
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