3110 −^4 –10−^5 per nucleotide, DNA repair pathways reduce replicative
error rates to 10−^9 –10−^11 per nucleotide [ 5 , 6 ]. Major contributor
to the DNA replication fidelity is mismatch repair (MMR), which
eliminates 99.99% of the errors generated by the replicative DNA
polymerases [ 6 ]. During replication, MMR detects DNA replica-
tion errors and recruits enzymes to destroy the portion of the
DNA strand that contains the error. MMR is found in all domains
of life and its function is evolutionarily highly conserved. To cor-
rect errors, MMR protein MutS binds to the sites of DNA replica-
tion errors and recruits MutL MMR protein. In enterobacteria,
the MutL bound to mismatch-MutS complex recruits MutH, an
endonuclease that cleaves the newly replicated DNA strand in the
proximity of the error. This triggers the removal of a segment of
single-stranded DNA containing the wrong base by the UvrD
DNA helicase and ssDNA exonucleases. The repair process is final-
ized by DNA polymerase III and DNA ligase activity. Eukaryotes
use homologs of MutS and MutL to correct errors in DNA replica-
tion, but lack a homolog of MutH [ 6 ].
We showed that MMR system can be exploited to visualize the
unrepaired DNA replication errors, i.e., emerging DNA muta-
tions. By imaging the fluorescently labeled components of MMR,
we found that fluorescent MutL protein forms foci on such DNA
sites [ 7 ], probably due to extensive accumulation of MutL proteins
when repair cannot be completed. Our conclusion that the fluores-
cent MutL foci tag emerging DNA mutations is based on two find-
ings. Previously, DNA sequencing data and fluctuation analysis
estimated the mutation rate of wild-type Escherichia coli to about
one mutation per 300 cells [ 8 , 9 ]. In good agreement with this, we
found that the frequency of fluorescent MutL foci in wild-type E.
coli cells is about 1 MutL foci per 230 cells [ 7 ] (Fig. 1a). Second,
we found that cells that mutate 50–1000-fold more compared to
wild-type E. coli cells show 50–1000-fold more MutL foci [ 7 ]
(Fig. 1b). We also showed that the mutation rates estimated by
fluctuation analyses are proportional to the MutL foci frequency
over a several hundred-fold range [ 7 ]. This real-time method for
mutation detection allows recording mutation rates of thousands
of individual cells in less than an hour. Furthermore, using this
method we could detect up to seven mutations per single cells as
they appear, before the expression of the mutation phenotype.
Finally, because MutL foci disappear from the sites of emerging
mutations when a new replication fork passes through, detecting
fluorescent MutL foci allows direct measuring of the per genera-
tion mutation rate. This finding was based on our data showing
that preventing new rounds of DNA replication by treating cells
with rifampicin prevents disappearance of the MutL foci.
Detecting Mutations in Living Cells