New complexities of SOS-induced “untargeted” mutagenesis in Escherichia coli as revealed by mutation accumulation and whole-genome sequencing

Abstract

When its DNA is damaged, Escherichia coli induces the SOS response, which consists of about 40 genes that encode activities to repair or tolerate the damage. Certain alleles of the major SOS-control genes, recA and lexA, cause constitutive expression of the response, resulting in an increase in spontaneous mutations. These mutations, historically called "untargeted", have been the subject of many previous studies. Here we re-examine SOS-induced mutagenesis using mutation accumulation followed by whole-genome sequencing (MA/WGS), which allows a detailed picture of the types of mutations induced as well as their sequence-specificity. Our results confirm previous findings that SOS expression specifically induces transversion base-pair substitutions, with rates averaging about 60-fold above wild-type levels. Surprisingly, the rates of G:C to C:G transversions, normally an extremely rare mutation, were induced an average of 160-fold above wild-type levels. The SOS-induced transversion showed strong sequence specificity, the most extreme of which was the G:C to C:G transversions, 60% of which occurred at the middle base of 5′GGC3′+5′GCC3′ sites although these sites represent only 8% of the G:C base pairs in the genome. SOS-induced transversions were also DNA strand biased, occurring, on average, 2-to 4-times more often when the purine was on the leading strand template and the pyrimidine on the lagging strand template than in the opposite orientation. However, the strand bias was also sequence specific, and even of reverse orientation at some sites. By eliminating constraints on the mutations that can be recovered, the MA/WGS protocol revealed new complexities to the nature of SOS "untargeted" mutations.

DNA Replication-Transcription Conflicts Do Not Significantly Contribute to Spontaneous Mutations Due to Replication Errors in Escherichia coli

“…To test if a similar mutational signature occurred in our experiments, we compared the frequencies of all six types of BPSs in CDSs in the CD and HO orientations ( Table 7 ). We included all six BPSs because (i) G·C-to-A·T transitions might be induced by replication-transcription conflicts but could not have been scored in the assay used by Million-Weaver et al ( 14 ) because a G·C-to-A·T change would create a TAA stop codon and (ii) E. coli ’s Y-family polymerases, Pol IV and Pol V, frequently create transversions ( 44 – 49 ). However, the frequencies of transversions are low, so we grouped them into A·T and G·C transversions for each gene.…”

CRISPR-Cas9-assisted genome editing inE. colielevates the frequency of unintended mutations

“…The observation that constitutively expressed Pol V can cause mutations in the absence of DNA damage (67) is likely due to its ability to substitute for Pol III at stalled replication forks, allowing translesion synthesis (68). Additionally, Pol V in association with the β clamp can fill in single-strand gaps left when the Pol III replisome skips over a DNA lesion (60, 69, 70).…”

Escherichia coli DNA replication: the old model organism still holds many surprises

“…Interestingly, in the recA730 genetic background, where RecA_E38K exists in a constitutively induced state due to the more efficient competition with SSB, most of the regulation is circumvented such that Pol V is constantly expressed without any DNA damage, promoting high levels of spontaneous mutagenesis, especially when combined with a LexA deficiency (e.g. lexA51 ) (Watanabe-Akanuma et al 1997 , Niccum et al 2020 ).…”