Seeing Mutations in Living Cells

Abstract

Summary Background Evolution depends on mutations: rare errors in the transmission of genetic information. Experimentally, mutations have been found by detecting altered phenotypes or sequencing complete genomes, but most mutations do not have overt phenotypes, and sequencing is expensive and has limited time resolution. The major source of mutations is DNA replication errors. Nearly all mistakes in DNA replication are detected and repaired by the mismatch repair machinery. Results We use a functional, fluorescently labeled derivative of one of the key mismatch repair proteins (MutL) to see and count the small fraction of errors in Escherichia coli that does not get repaired and is converted into stable mutations by the next round of DNA replication. Over a 300-fold range, there is a linear relationship between the frequency of fluorescent foci and the genetically measured mutation frequency, and the mean frequency of fluorescent foci agrees well with estimates of the global mutation rate. Conclusion We describe a method for detecting the majority of genomic mutations emerging in living cells, independently of their potential phenotype. The distribution of emerging mutations per cell is roughly Poisson distributed, suggesting that all the cells in the population have roughly the same mutation rate.

New insights into the mechanism of DNA mismatch repair

“…MMR complexes have been visualized in bacteria and in human cell lines (Elez et al 2010; Hong et al 2008; Kleczkowska et al 2001; Roesner et al 2013; Smith et al 2001). However, in these studies, the use of overexpression systems has complicated the interpretation of the results.…”

Stochastic activation of a DNA damage response causes cell-to-cell mutation rate variation

“…We therefore tested whether heterogeneity in Ada concentrations affects mutation rates. To directly measure genomic mutation rates in single cells we used the DNA mismatch recognition protein MutS as a marker for labelling nascent mutations (22). Specifically, photoactivated single-molecule tracking (23) allowed us to classify individual MutS-PAmCherry fusion proteins as DNA-bound or mobile (24, 25), while also imaging Ada-mYPet in the same live cells (Fig.…”

Postreplicative Mismatch Repair

“…The formation of these foci was dependent on the ability of MutS to interact with b (Simmons et al 2008). In E. coli, a similar strategy was deployed to visualize nascent mutations, that is, mispairs that escaped repair after replication and that persisted until the following round of replication (Elez et al 2010). Interestingly, the foci that persisted were those generated by MutL rather than MutS.…”