It is well known that exposure of cells to heat leads to a drastic inhibition of DNA synthesis as assayed in vivo by the incorporation of radioactive precursors into acid-insoluble material. Here we introduce an SV40 in vitro DNA replication assay and show that this inhibition may be partly due to the activation of a checkpoint in S phase that stalls the initiation of DNA replication by inactivating replication protein A (RPA), an essential factor for replication. The results implicate trans-acting processes in the regulation of DNA replication after heat exposure and suggest that such processes may be an integral part of the normal response to heat insult. The observations extend and complement previous studies that have implicated heat-induced chromatin damage acting in cis as a cause for the observed inhibition of DNA synthesis in cells exposed to hyperthermia. A model is proposed postulating that the presence of single-stranded DNA, or heat-induced damage to chromatin structures directly, albeit passively, inhibits the elongation stages of ongoing DNA replication. It is hypothesized that arrested replication forks subsequently act as signals to activate the S-phase checkpoint that actively inhibits the initiation of new replicons. The ultimate purpose of this response will be the minimization of the toxic consequences of heat-induced damage, as it may delay DNA replication until chromatin conformation has been restored. DNA replication in the presence of chromatin damage has been implicated in the formation of lethal chromosome aberrations observed in cells heated during S phase. The operation of active processes in the regulation of DNA replication in cells exposed to hyperthermia offers new targets for intervention and sensitization of cells to heat.

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