We are interested in determining the mechanisms by which human cells control spontaneous and induced mutation rates. We have previously shown that the major replicative DNA polymerase in human cells, when purified as a relatively simple DNA polymerase of limited complexity, is not accurate enough during in vitro DNA synthesis reactions to account for low spontaneous mutations rates in vivo. However, it is well established that replication of DNA involves the concerted action of a number of proteins interacting as a complex """"""""replisome"""""""". We therefore want to examine the fidelity of actual semiconservative bidirectional replication by this complex protein machine, in a way in which we can ultimately dissect the importance of individual proteins and their interactions to specific mutational pathways and molecular mechanisms. To do this we are using a mammalian viral model system of human DNA replication in combination with our recently developed, highly sensitive genetic assay to monitor mutagenesis. Our initial results suggest that this SV40 replication system is highly accurate, exhibiting more than 20-fold higher fidelity than the purified replicative DNA polymerase alone. The effect could be much greater, since our current measurements are limited by the background mutation frequency of the first assay we have employed. This higher fidelity could result from any of several steps in discrimination of the error rate of replication using more sensitive assays. We will then focus on mechanisms by fractionating and defining the contributions to fidelity of individual protein components and by using specifically engineered template DNA molecules as mutational targets.
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