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The frequency of MMS-induced, umuDC-dependent, mutations declines during starvation in Escherichia coli
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw.
Abstract:
It has been found that the level of methyl methanesulfonate (MMS)-induced mutation in Escherichia coli is dependent on the level of UmuD(D')C proteins. The frequency of argE(ochre)-->Arg+ mutations (which occur predominantly by AT-->TA transversions) and RifS-->RifR mutations is much higher when UmuDC or UmuD'C are overproduced in the cell. When MMS-treated bacteria were starved for progressively longer times and hence the expression of mutations delayed, the level of mutations observed progressively declined. This same treatment had no effect on the degree of SOS induction. Examination of plasmid DNAs, isolated from MMS-treated cells, for their sensitivity to the specific endonucleases Fpg and Nth revealed that MMS causes formation of abasic sites, which are repaired during cell starvation. It is assumed that, in non-dividing cells, apurinic sites are mostly repaired by RecA-mediated recombinational repair. This pathway, which is error-free, is compared with the processing pathway in metabolically active cells, where translesion synthesis by the UmuD'2C-RecA-DNA polymerase III holoenzyme complex occurs; this latter pathway is error-prone.
Insights
Methyl methanesulfonate (MMS) exposure in Escherichia coli increases mutation frequency, particularly when UmuD(D′)C proteins are overproduced. Error-free repair pathways dominate in non-dividing cells, contrasting with error-prone translesion synthesis in active cells.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Methyl methanesulfonate (MMS) is a mutagen that induces DNA damage.
- The UmuD(D′)C protein complex plays a crucial role in DNA repair and mutagenesis.
- Escherichia coli possesses distinct DNA repair mechanisms, including SOS response and translesion synthesis.
Purpose of the Study:
- To investigate the relationship between UmuD(D′)C protein levels and MMS-induced mutation frequency in Escherichia coli.
- To elucidate the role of cell metabolic state (dividing vs. non-dividing) in DNA repair pathways following MMS exposure.
- To compare error-free and error-prone DNA repair mechanisms in response to MMS-induced DNA lesions.
Main Methods:
- Overproduction of UmuD(D′)C proteins in Escherichia coli.
- Treatment of bacteria with methyl methanesulfonate (MMS).
- Mutation frequency analysis for argE(ochre) and RifS-->RifR mutations.
- Cell starvation experiments to delay mutation expression.
- Plasmid DNA isolation and sensitivity testing with Fpg and Nth endonucleases.
- Assessment of SOS induction levels.
Main Results:
- MMS-induced mutation frequency, specifically AT-->TA transversions, is significantly higher with UmuD(D′)C overproduction.
- Delayed mutation expression during cell starvation leads to a progressive decline in observed mutations.
- MMS induces abasic sites in DNA, which are repaired during cell starvation.
- SOS induction is not affected by cell starvation treatment.
- RecA-mediated recombinational repair, an error-free pathway, is implicated in non-dividing cells.
Conclusions:
- The level of UmuD(D′)C proteins dictates the frequency of MMS-induced mutations in Escherichia coli.
- Cellular metabolic state influences the predominant DNA repair pathway, with error-free repair favored in non-dividing cells.
- Translesion synthesis by the UmuD'2C-RecA-DNA polymerase III holoenzyme complex represents an error-prone pathway active in metabolically active cells.