Base Pair Hydrogen Bonds Are Essential for Proofreading Selectivity by the Human Mitochondrial DNA Polymerase
| Autor: | Kenneth A. Johnson, Sandra A. Helquist, Eric T. Kool, Harold R. Lee |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Exonuclease
Steric effects Base pair Stereochemistry DNA-Directed DNA Polymerase Biochemistry Nucleic acid secondary structure Humans Nucleotide Base Pairing Molecular Biology Polymerase chemistry.chemical_classification Biological Products Molecular Structure biology Nucleotides Chemistry Hydrogen bond Hydrogen Bonding Cell Biology DNA Polymerase gamma Kinetics DNA: Replication Repair Recombination and Chromosome Dynamics biology.protein Proofreading |
| Zdroj: | Journal of Biological Chemistry. 283:14411-14416 |
| ISSN: | 0021-9258 |
| Popis: | We have characterized the role of Watson-Crick hydrogen bonding in the 3'-terminal base pair on the 3'-5' exonuclease activity of the human mitochondrial DNA polymerase. Nonpolar nucleoside analogs of thymidine (dF) and deoxyadenosine (dQ) were used to eliminate hydrogen bonds while maintaining base pair size and shape. Exonuclease reactions were examined using pre-steady state kinetic methods. The time dependence of removal of natural nucleotides from the primer terminus paired opposite the nonpolar analogs dF and dQ were best fit to a double exponential function. The double exponential kinetics as well as the rates of excision (3-6 s(-1) fast phase, 0.16-0.3 s(-1) slow phase) are comparable with those observed during mismatch removal of natural nucleotides even when the analog was involved in a sterically correct base pair. Additionally, incorporation of the next correct base beyond a nonpolar analog was slow (0.04-0.22 s(-1)), so that more than 95% of terminal base pairs were removed rather than extended. The polymerase responds to all 3'-terminal base pairs containing a nonpolar analog as if it were a mismatch regardless of the identity of the paired base, and kinetic partitioning between polymerase and exonuclease sites failed to discriminate between correct and incorrect base pairs. Thus, sterics alone are insufficient, whereas hydrogen bond formation is essential for proper proofreading selectivity by the mitochondrial polymerase. The enzyme may use the alignment and prevention of fraying provided by proper hydrogen bonding and minor groove hydrogen bonding interactions as critical criteria for correct base pair recognition. |
| Databáze: | OpenAIRE |
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