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A coproofreading Zn(2+)-dependent exonuclease within a bacterial replicase.
Natalie M Stano1, Joe Chen, Charles S McHenry
1Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Nature Structural & Molecular Biology
|April 11, 2006
Summary
DNA polymerase proofreading involves a novel Zn(2+)-dependent exonuclease within the alpha subunit
Area of Science:
- Molecular Biology
- Enzymology
- DNA Replication
Background:
- DNA replicases utilize proofreading exonucleases with acidic residues chelating Mg(2+) ions for catalysis.
- DNA polymerase III holoenzymes possess proofreading activity in the epsilon subunit, interacting with the alpha subunit via its N-terminal php domain.
Purpose of the Study:
- To investigate the functional role of the alpha subunit's N-terminal php domain in DNA polymerase III holoenzymes.
- To identify and characterize novel exonuclease activities associated with DNA replication machinery.
Main Methods:
- Biochemical assays to detect and characterize nuclease activity.
- Site-directed mutagenesis to probe the function of the alpha php domain.
- Enzyme kinetics to determine substrate specificity and cofactor requirements.
Main Results:
- Demonstrated a novel Zn(2+)-dependent 3' --> 5' exonuclease activity within the alpha php domain.
- This coediting nuclease preferentially removes mispaired nucleotides during DNA synthesis.
- Established the alpha php domain as a distinct proofreading component.
Conclusions:
- The alpha php domain harbors a unique coediting nuclease, expanding the known mechanisms of DNA replication fidelity.
- This finding provides the first example of a coediting nuclease functioning within the polymerase subunit itself.