Removal of 8-oxo-GTP by MutT hydrolase is not a major contributor to transcriptional fidelity.
Bottom Line: We do not observe any increase in epigenetic switching in mutT cells.Moreover, we observe a fluctuation type of distribution of β-galactosidase appearance in a growing culture, consistent with Lac+ DNA revertant events.We conclude that the absence of MutT produces a DNA mutator but does not equally create an RNA mutator.
Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.Show MeSH
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Mentions: To qualitatively monitor β-galactosidase enzyme activity in growing cultures, Xgal (0.5 mg/ml) was added to LB media (39), and a fluctuation test was performed. We seeded ∼200 cells to wells of a microtiter dish and monitored growth (OD600) and cleavage of Xgal (OD615) to observe the nature of the appearance of β-galactosidase activity during growth of the CC101 and ΔmutT compromised cultures (Figure 5A). As the burden of mutT-mediated mutagenesis is attenuated from ΔmutT to ΔmutT ΔmutY or ΔmutT dnaE941 to ΔmutT ΔmutY dnaE941, the resulting cultures become less uniformly blue, and only some cultures turn blue whereas other cultures of the same strain remain clear. Indeed, in the CC101 ΔmutT ΔmutY dnaE941 strain, the great majority of wells were clear and similar in appearance to the wild-type parent CC101 (as shown in the OD615 tracings; Figure 5A bottom panel). Therefore, although the amounts of 8-oxo-dGTP and 8-oxo-GTP remain the same in all these ΔmutT strains, the phenotypic consequences of these oxidized nucleotides are decreased; we emphasize that it is only the mutational consequences of 8-oxo-dGTP that are precluded, since DNA polymerase III and MutY do not incorporate 8-oxo-GTP nor act on 8-oxo-GTP incorporation, respectively.
Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.