DNA Replication Errors Produced by the Replicative Apparatus of Escherichia coli

Author： Fujii S. Akiyama M. Aoki K. Sugaya Y. Higuchi K. Hiraoka M. Miki Y. Saitoh N. Yoshiyama K. Ihara K. Seki M. Ohtsubo E. Maki H.

Publisher： Academic Press

ISSN： 0022-2836

Source： Journal of Molecular Biology, Vol.289, Iss.4, 1999-06, pp. : 835-850

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Abstract

It has been hard to detect forward mutations generated during DNA synthesis in vitro by replicative DNA polymerases, because of their extremely high fidelity and a high background level of pre-existing mutations in the single-stranded template DNA used. Using the oriC plasmid DNA replication in vitro system and the rpsL forward mutation assay, we examined the fidelity of DNA replication catalyzed by the replicative apparatus of Escherichia coli . Upon DNA synthesis by the fully reconstituted system, the frequency of rpsL^-mutations in the product DNA was increased to 1.9×10^-4, 50-fold higher than the background level of the template DNA. Among the mutations generated in vitro , single-base frameshifts predominated and occurred with a pattern similar to those induced in mismatch-repair deficient E. coli cells, indicating that the major replication error was slippage at runs of the same nucleotide. Large deletions and other structural alterations of DNA appeared to be induced also during the action of the replicative apparatus.