Highly mutagenic replication by DNA polymerase V (UmuC) provides a mechanistic basis for SOS untarqeted mutagenesis

摘要

When challenged by DNA-damaging agents, EScherichia coIi cells respond by inducing the SOS stress response, which leads to an increase in mutation frequency by two mechanisms: translesion replication, a process that causes mutations because of misinser- tion opposite the lesions, and an inducible mutator activity. which acts at undamaged sites. Here we report that DNA polymerase V (pol V; UmuC), which previously has been shown to be a lesion- bypass DNA polymerase. was highly mutagenic during in vitro gap-filling replication of a gapped plasmid carrying the cro re- porter gene. This reaction required, in addition to poI V, UmuD'. Reot, and single-stranded DNA (ssDNA)-binding protein. poI V produced point mutations at a frequency of 2.1 x 10~-4 per nucleotide (2.1/100 per cro gene). 41-fold higher than DNA polymer- ase lll holoenzyme. The mutational spectrum of pol V was domi- nated by transversions (53/100), which were formed at a frequency of 1.3 x 10~4 per nucleotide (1.1/100 per cro gene), 74-fold higher than with pol lll holoenzyme. The prevalence of transversions and the protein requirements of this system are similar to those of in vivo untargeted mutagenesis (SOS mutator activity). This finding suggests that replication by pol V, in the presence of UmuD', RecA, and ssDNA-binding protein, is the basis of chromosomal SOS untargeted mutagenesis.