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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Mutational consequences of dNTP pool imbalances in E. coli
Roel M Schaaper1, Christopher K Mathews
1Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, 111 TW Alexander Drive, Research Triangle Park, NC 27709, USA. schaaper@niehs.nih.gov
DNA Repair
|December 11, 2012
Summary
Altering deoxynucleoside triphosphate (dNTP) pools in Escherichia coli impacts DNA synthesis accuracy. Specific dNTP pool imbalances in dcd and ndk strains correlate with distinct mutation patterns, revealing in vivo replication error mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- DNA synthesis accuracy relies on polymerase fidelity and deoxynucleoside triphosphate (dNTP) precursor availability.
- In vivo relationships between dNTP pools and replication error rates remain incompletely understood.
Purpose of the Study:
- Investigate the in vivo impact of altered dNTP pools on DNA replication fidelity in Escherichia coli.
- Analyze the mutator phenotypes of dcd and ndk strains, which exhibit dNTP pool disturbances.
Main Methods:
- Generated and analyzed dcd and ndk mutant strains of Escherichia coli.
- Quantified intracellular dNTP pool levels (dCTP, dGTP, dATP).
- Assessed mutation rates using lacZ reversion markers in a mismatch-repair defective (mutL) background and rifampicin resistance assays.
Main Results:
- dcd strains showed increased dCTP and decreased dGTP, correlating with enhanced G·C→T·A and A·T→T·A transversions.
- ndk strains exhibited decreased dATP and slightly increased dCTP, specifically enhancing A·T→T·A transversions.
- Both strains displayed differential mutability for rifampicin-resistant mutants, consistent with dNTP pool alterations.
Conclusions:
- Demonstrated a satisfactory correlation between dNTP pool imbalances and replication error rates in vivo.
- The mutator phenotype of the ndk strain can be minimally explained by predicted dNTP pool effects.
- Provides insights into the mechanisms linking dNTP availability to DNA replication fidelity.
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