Spontaneous Hotspot Mutations Resistant to Mismatch Correction in Escherichia coli: Transcription-dependent Mutagenesis Involving Template-switching Mechanisms

The generation and stabilization of spontaneous mutations are affected by many factors, including the accuracy of DNA replication, the generation of spontaneous DNA lesions, and the capacity of mutation-avoidance systems. However, little is known about the causes of spontaneous mutations in cells with fully active mutation-avoidance systems. Using the rpsL forward mutation assay, we previously found that the directionality of replication fork movement significantly affects spontaneous mutagenesis in Escherichia coli. In particular, sequence substitutions and a hotspot type of single-base frameshift, both of which are caused by quasipalindrome-directed mutagenesis, appeared to depend on the directionality of the replication fork. These mutations are also resistant to post-replicative mismatch correction. Here, we show that the level of transcription of the rpsL gene strongly affects spontaneous mutagenesis at two mutational hotspot sites in the target sequence, one for a T→G base substitution and the other for a+1 single-base frameshift. Mutation frequencies at the hotspot sites were below a detectable level when the transcription of the target sequence was tightly suppressed, but were dramatically increased when the target sequence was highly transcribed. Both of the hotspot mutations were also dependent on the directionality of the replication fork and were caused by quasipalindrome-directed mutagenesis. The frequencies of the hotspot mutations were unchanged in a mismatch-repair deficient strain, indicating that the hotspot mutations are resistant to the mismatch correction. Based on these findings, we propose a novel mutagenic process for these hotspot mutations that depends on transcription and involves template-switching mechanisms induced by spontaneous DNA lesions.