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The E249K mutator mutant of DNA polymerase beta extends mispaired termini
1Yale University School of Medicine, Department of Therapeutic Radiology, New Haven, Connecticut 06520, USA.
The Journal of Biological Chemistry
|December 10, 1999
Summary
A DNA polymerase beta mutant (E249K) shows a mutator phenotype not due to base misincorporation. Instead, it efficiently extends mismatched DNA bases, revealing Glu-249
Area of Science:
- Molecular Biology
- Enzymology
- Genetics
Background:
- DNA polymerase beta (pol beta) is crucial for DNA repair.
- Mutations in pol beta can lead to a mutator phenotype, increasing mutation rates.
- The E249K mutation in pol beta alters its fidelity.
Purpose of the Study:
- Investigate the basis of the mutator phenotype in the E249K pol beta mutant.
- Determine if the mutation affects nucleotide misincorporation or mispair extension fidelity.
- Elucidate the role of the Glu-249 residue in pol beta fidelity.
Main Methods:
- Primer extension assays to measure misincorporation fidelity.
- Single turnover conditions to analyze kinetic mechanisms.
- Missing base primer extension assays to assess mispair extension.
- Comparison of E249K mutant with wild-type pol beta.
Main Results:
- The E249K mutant showed no significant difference in misincorporation fidelity compared to wild-type.
- The mutation altered nucleotide discrimination, favoring initial binding over conformational/chemical steps.
- The E249K mutant exhibited enhanced extension of mispaired primer termini (G:A and C:T).
Conclusions:
- The mutator phenotype of E249K pol beta results from an increased ability to extend mispaired DNA bases, not misincorporation.
- The Glu-249 side chain is critical for maintaining fidelity against mispair extension.
- Altered kinetic mechanisms of discrimination contribute to the observed fidelity defects.