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Abstract

Mercuric ion is cytotoxic and mutagenic to cells. However, the mechanisms of mercuric ion-induced cytotoxicity are not well understood. Studies have suggested that these effects may be due in part to the alteration and inhibition of a variety of cellular processes including DNA replication, DNA repair, RNA transcription, and protein synthesis. However, prior art studies utilizing whole cells, cell extracts, or purified DNA polymerases to examine these activities are not able to specifically identify the precise mechanism or site of the effect or adequately represent the highly ordered environment in which DNA replication occurs in the intact cell. We disclose a novel method for measuring the activity and fidelity of DNA replication using the complex of proteins called the DNA synthesome, an isolated multiprotein form of DNA polymerase. The DNA synthesome is a highly organized complex of proteins capable of supporting all phases of SV 40 origin-specific DNA replication in vitro. The assay and kit described herein can be used both to examine the mechanism of cytotoxicity and mutagenicity and to identify compounds that alter the activity and fidelity of the DNA synthetic apparatus such as heavy metal ion, particularly mercuric ion. The assay and kit can be used to rapidly screen compounds for toxic activity.

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