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Infidelity of DNA synthesis as a cause of mutagenesis
Abstract:
The concept underlying these studies is that a major determinant of mutagenesis involves perturbations in the fidelity of DNA replication. i.e., the accuracy by which DNA polymerases copy DNA templates. To investigate this relationship, we have designed in vitro assays to measure the accuracy of DNA replication and used these systems to screen for and to quantitate factors that promote errors in DNA synthesis. Using DNA polymerase from bacteria, the frequency of mistakes with phi X174 DNA as a template approaches 10(-7) and is similar to the spontaneous mutation rates in bacterial cells. In contrast, DNA polymerases from animal cells are more error-prone. The differences in fidelity among mammalian DNA polymerases which lack error-correcting mechanisms suggest that these enzymes enhance accuracy by improving base-selection. Thus, mutants in DNA polymerase-alpha might be altered in base-selection. Chinese hamster V79 cell mutants selected by resistance to aphidicolin, a specific inhibitor of DNA polymerase-alpha, have been reported (Somatic Cell Genet., 7: 235-253, 1981). DNA polymerase-alpha was purified from mitochondria-free crude extracts of these mutants by sequential column chromatography using DEAE-cellulose and phosphocellulose. DNA polymerase-alpha purified from one of the mutants is 10-fold more resistant to aphidicolin than the same enzyme purified from the parental cells. Moreover, the apparent Km for dCTP is 1.0 +/- 0.4 microM for the mutant polymerase and 10 +/- 4 microM for the parental enzyme. These observed differences are in accord with the known competition between aphidicolin and dCTP, and provide a mechanism for the aphidicolin resistance of the mutant, i.e., the decrease in Km for dCTP. The elevated spontaneous and induced mutation rate exhibited by this mutant could be mediated by the alteration in DNA polymerase-alpha. With DNA replicating enzymes from a variety of sources, enhancement of mutagenesis has been demonstrated by alteration in precursor pools, damage to DNA templates, loss of nucleotide bases on DNA, metal ions that interact with nucleotide bases, and organic compounds that intercalate into DNA. The alterations of deoxynucleoside triphosphate pools also occur after treatment of animal cells with known mutagens. This observation may provide a new mechanism for mutagenesis by these agents independent of alterations in DNA.
Insights
DNA replication fidelity is crucial for preventing mutations. Animal DNA polymerases are less accurate than bacterial ones, and this study investigates a mutant DNA polymerase-alpha with altered base selection, potentially explaining increased mutation rates.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA replication fidelity, the accuracy of DNA polymerases, is a key factor in mutagenesis.
- Animal DNA polymerases are generally more error-prone than bacterial counterparts, lacking efficient error-correction mechanisms.
- Mutations in DNA polymerase-alpha may affect base selection, influencing DNA synthesis accuracy.
Purpose of the Study:
- To investigate the relationship between DNA replication fidelity and mutagenesis.
- To screen for and quantify factors that promote errors during DNA synthesis.
- To characterize aphidicolin-resistant Chinese hamster V79 cell mutants with potential alterations in DNA polymerase-alpha.
Main Methods:
- Development of in vitro assays to measure DNA replication accuracy.
- Purification of DNA polymerase-alpha from mutant and parental Chinese hamster V79 cell extracts using sequential column chromatography (DEAE-cellulose and phosphocellulose).
- Biochemical characterization of purified DNA polymerase-alpha, including aphidicolin resistance and Michaelis-Menten kinetics (Km for dCTP).
Main Results:
- Bacterial DNA polymerase fidelity approaches 10(-7) errors, similar to bacterial spontaneous mutation rates.
- DNA polymerase-alpha purified from an aphidicolin-resistant mutant showed 10-fold higher resistance to aphidicolin compared to the parental enzyme.
- The mutant DNA polymerase-alpha exhibited a significantly lower apparent Km for dCTP (1.0 +/- 0.4 microM) compared to the parental enzyme (10 +/- 4 microM), indicating altered base selection.
Conclusions:
- The observed decrease in Km for dCTP in the mutant DNA polymerase-alpha provides a mechanism for aphidicolin resistance.
- Alterations in DNA polymerase-alpha, specifically in base selection, may mediate the elevated spontaneous and induced mutation rates in the mutant.
- Changes in deoxynucleoside triphosphate pools can occur after exposure to mutagens, potentially offering a novel mutagenesis mechanism independent of direct DNA alterations.