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Removal of 8-oxo-GTP by MutT hydrolase is not a major contributor to transcriptional fidelity.

Gordon AJ, Satory D, Wang M, Halliday JA, Golding I, Herman C - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

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A fluctuation test analysis of Lac+ revertants in CC101 and ΔmutT compromised strains. Overnight LB cultures of each strain were diluted and ∼200 cells were seeded into fresh LB media containing 0.5 mg/ml Xgal in a microtiter dish (200 μl per well) and grown at 37°C with shaking in an automated plate reader. Readings were recorded every hour for 46 h (OD600 to monitor cell growth; OD615 to monitor Xgal cleavage). (A) False color microtiter plate after 46 h growth. Rows 1 and 2, CC101; rows 2 and 3, CC101 ΔmutT; row 5, CC101 ΔmutT ΔmutY; row 6, CC101 ΔmutT dnaE941; rows 7 and 8, CC101 ΔmutT ΔmutY dnaE941. The first column is an LB control (cells but no Xgal; OD600 readings from these wells were subtracted from OD615 readings from wells containing the same strain with Xgal to normalize readings to account for cell growth as shown in (B)); all other wells contain LB plus Xgal. The corresponding OD615 traces are found beneath the microtiter plate. (B) Heat map representation of the OD615 scans over time showing the fluctuation nature of the observance of Lac+ mutation. Each row represents normalized hourly OD615 readings starting at hour 9 and ending at hour 46. First panel shows CC101 results from row 1 in (A); second panel shows CC101 ΔmutT results from row 3 in (A); third panel shows CC101 ΔmutT dnaE941 results from row 6 in (A); fourth panel shows CC101 ΔmutT ΔmutY results from row 5 in (A); last panel shows CC101 ΔmutT ΔmutY dnaE941 from row 7 in (A).
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Figure 5: A fluctuation test analysis of Lac+ revertants in CC101 and ΔmutT compromised strains. Overnight LB cultures of each strain were diluted and ∼200 cells were seeded into fresh LB media containing 0.5 mg/ml Xgal in a microtiter dish (200 μl per well) and grown at 37°C with shaking in an automated plate reader. Readings were recorded every hour for 46 h (OD600 to monitor cell growth; OD615 to monitor Xgal cleavage). (A) False color microtiter plate after 46 h growth. Rows 1 and 2, CC101; rows 2 and 3, CC101 ΔmutT; row 5, CC101 ΔmutT ΔmutY; row 6, CC101 ΔmutT dnaE941; rows 7 and 8, CC101 ΔmutT ΔmutY dnaE941. The first column is an LB control (cells but no Xgal; OD600 readings from these wells were subtracted from OD615 readings from wells containing the same strain with Xgal to normalize readings to account for cell growth as shown in (B)); all other wells contain LB plus Xgal. The corresponding OD615 traces are found beneath the microtiter plate. (B) Heat map representation of the OD615 scans over time showing the fluctuation nature of the observance of Lac+ mutation. Each row represents normalized hourly OD615 readings starting at hour 9 and ending at hour 46. First panel shows CC101 results from row 1 in (A); second panel shows CC101 ΔmutT results from row 3 in (A); third panel shows CC101 ΔmutT dnaE941 results from row 6 in (A); fourth panel shows CC101 ΔmutT ΔmutY results from row 5 in (A); last panel shows CC101 ΔmutT ΔmutY dnaE941 from row 7 in (A).

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.


Removal of 8-oxo-GTP by MutT hydrolase is not a major contributor to transcriptional fidelity.

Gordon AJ, Satory D, Wang M, Halliday JA, Golding I, Herman C - Nucleic Acids Res. (2014)

A fluctuation test analysis of Lac+ revertants in CC101 and ΔmutT compromised strains. Overnight LB cultures of each strain were diluted and ∼200 cells were seeded into fresh LB media containing 0.5 mg/ml Xgal in a microtiter dish (200 μl per well) and grown at 37°C with shaking in an automated plate reader. Readings were recorded every hour for 46 h (OD600 to monitor cell growth; OD615 to monitor Xgal cleavage). (A) False color microtiter plate after 46 h growth. Rows 1 and 2, CC101; rows 2 and 3, CC101 ΔmutT; row 5, CC101 ΔmutT ΔmutY; row 6, CC101 ΔmutT dnaE941; rows 7 and 8, CC101 ΔmutT ΔmutY dnaE941. The first column is an LB control (cells but no Xgal; OD600 readings from these wells were subtracted from OD615 readings from wells containing the same strain with Xgal to normalize readings to account for cell growth as shown in (B)); all other wells contain LB plus Xgal. The corresponding OD615 traces are found beneath the microtiter plate. (B) Heat map representation of the OD615 scans over time showing the fluctuation nature of the observance of Lac+ mutation. Each row represents normalized hourly OD615 readings starting at hour 9 and ending at hour 46. First panel shows CC101 results from row 1 in (A); second panel shows CC101 ΔmutT results from row 3 in (A); third panel shows CC101 ΔmutT dnaE941 results from row 6 in (A); fourth panel shows CC101 ΔmutT ΔmutY results from row 5 in (A); last panel shows CC101 ΔmutT ΔmutY dnaE941 from row 7 in (A).
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Figure 5: A fluctuation test analysis of Lac+ revertants in CC101 and ΔmutT compromised strains. Overnight LB cultures of each strain were diluted and ∼200 cells were seeded into fresh LB media containing 0.5 mg/ml Xgal in a microtiter dish (200 μl per well) and grown at 37°C with shaking in an automated plate reader. Readings were recorded every hour for 46 h (OD600 to monitor cell growth; OD615 to monitor Xgal cleavage). (A) False color microtiter plate after 46 h growth. Rows 1 and 2, CC101; rows 2 and 3, CC101 ΔmutT; row 5, CC101 ΔmutT ΔmutY; row 6, CC101 ΔmutT dnaE941; rows 7 and 8, CC101 ΔmutT ΔmutY dnaE941. The first column is an LB control (cells but no Xgal; OD600 readings from these wells were subtracted from OD615 readings from wells containing the same strain with Xgal to normalize readings to account for cell growth as shown in (B)); all other wells contain LB plus Xgal. The corresponding OD615 traces are found beneath the microtiter plate. (B) Heat map representation of the OD615 scans over time showing the fluctuation nature of the observance of Lac+ mutation. Each row represents normalized hourly OD615 readings starting at hour 9 and ending at hour 46. First panel shows CC101 results from row 1 in (A); second panel shows CC101 ΔmutT results from row 3 in (A); third panel shows CC101 ΔmutT dnaE941 results from row 6 in (A); fourth panel shows CC101 ΔmutT ΔmutY results from row 5 in (A); last panel shows CC101 ΔmutT ΔmutY dnaE941 from row 7 in (A).
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

Show MeSH
Related in: MedlinePlus