Method for Detecting and Studying Genome-Wide Mutations in Single Living Cells in Real Time

Abstract

DNA sequencing and fluctuation test have been choice methods for studying DNA mutations for decades. Although invaluable tools allowing many important discoveries on mutations, they are both highly influenced by fitness effects of mutations, and therefore suffer several limits. Fluctuation test is for example limited to mutations that produce an identifiable phenotype, which is the minority of all generated mutations. Genome-wide extrapolations using this method are therefore difficult. DNA sequencing detects almost all DNA mutations in population of cells. However, the obtained population mutation spectrum is biased because of the fitness effects of newly generated mutations. For example, mutations that affect fitness strongly and negatively are underrepresented, while those with a strong positive effect are overrepresented. Single-cell genome sequencing can solve this problem. However, sufficient amount of DNA for this approach is obtained by massive whole-genome amplification, which produces many artifacts. We describe the first direct method for monitoring genome-wide mutations in living cells independently of their effect on fitness. This method is based on the following three facts. First, DNA replication errors are the major source of DNA mutations. Second, these errors are the target for an evolutionarily conserved mismatch repair (MMR) system, which repairs the vast majority of replication errors. Third, we recently showed that the fluorescently labeled MMR protein MutL forms fluorescent foci on unrepaired replication errors. If not repaired, the new round of DNA synthesis fixes these errors in the genome permanently, i.e., they become mutations. Therefore, visualizing foci of the fluorescently tagged MutL in individual living cells allows detecting mutations as they appear, before the expression of the phenotype.