This laboratory has been interested in the effects of selenium compounds on nucleic acid synthesis. We have carried out an investigation of the effects of sodium selenite on DNA synthesis in 3 systems: intact cells, isolated nuclei, and extracted polymerases. We found that in intact cells as well as in isolated nuclei, selenite inhibits DNA synthesis significantly. However, we also found that selenite was relatively ineffective in inhibiting purified DNA polymerase, the enzyme which catalyzes most of the synthesis in isolated nuclei. In more recent experiments we have found that selenite can in fact inhibit DNA polymerase, if a sulfhydryl compound such as mercaptoethanol is present in the reaction. We have further found that other sulfhydryl compounds can have the same potentiating effect on the inhibition of DNA polymerase by selenite, although with varying potencies. We have also obtained evidence for a similar effect with another enzyme, RNA polymerase II from wheat germ. The overall objective of the project is to characterize this phenomenon, and to relate it to the inhibition by selenite of cellular DNA synthesis in intact cells and isolated nuclei. In order to accomplish this we will first characterize the specificity of the effect in terms of the SH compound by carrying out a series of dose-response experiments to generate a matrix of data on the effects of sodium selenite on DNA polymerase in the presence of a series of SH compounds. This data will provide information on the relative potencies of the various SH compounds and may provide information on structure-function relationships. We will then examine the specificity of the phenomenon in terms of the enzyme, by investigating the ability of selenite to inhibit several other DNA and RNA polymerases, in the presence of various SH compounds. From these results we will be able to determine whether there are differences between the enzymes in the relative potencies of the various SH compounds. We will investigate the mechanism of the potentiation by exploring three models for the interaction of SH compounds, selenite and the polymerase. We will also attempt to determine whether the inhibitory effect of selenite on DNA synthesis in isolated nuclei results from SH-potentiated inhibition of the DNA polymerase by selenite, by investigating the effect of adding SH compounds to the nuclei reactions, or depleting the nuclei of possible endogenous SH compounds.
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