DNA replication errors produced by the replicative apparatus of Escherichia coli

It has been hard to detect forward mutations generated during DNA synthesis in vitro by replicative DNA polymerases, because of their extremely high f idelity and a high background level of pre-existing mutations in the single -stranded template DNA used. Using the oriC plasmid DNA replication in vitr o system and the rpsL forward mutation assay, we examined the fidelity of D NA 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 x 10(-4), 50-fold hig her than the background level of the template DNA. Among the mutations gene rated in vitro, single-base frameshifts predominated and occurred with a pa ttern similar to those induced in mismatch-repair deficient E. coli cells, indicating that the major replication error was slippage at runs of the sam e nucleotide. Large deletions and other structural alterations of DNA appea red to be induced also during the action of the replicative apparatus. (C) 1999 Academic Press.