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Molecular mechanisms of manganese mutagenesis
Abstract:
The mechanism by which DNA polymerase discriminates between complementary and noncomplementary nucleotides for insertion into a primer terminus has been investigated. Apparent kinetic constants for the insertion of dGTP and dATP into the hook polymer d(C)194-d(G)12 with Escherichia coli DNA polymerase I (large fragment) were determined. The results suggest that the high specificity of base selection by DNA polymerase I is achieved by utilization of both Km and Vmax differences between complementary and noncomplementary nucleotides. The molecular basis for the increased error frequency observed with DNA polymerase I in the presence of Mn2+ has also been investigated. Our studies demonstrate that when Mn2+ is substituted for Mg2+, there is a higher ratio of insertion of incorrect to correct dNTP by the polymerase activity, accompanied by a decreased hydrolysis of a mismatched dNMP relative to a matched dNMP at the primer terminus by the 3',5' exonuclease activity. Kinetic analysis revealed that in the presence of Mn2+, the kcat for insertion of a complementary dNTP is reduced, whereas the catalytic rate for the insertion of a mismatched nucleotide is increased. The apparent Km values for either complementary or noncomplementary nucleotide substrates are not significantly altered when Mg2+ is replaced by Mn2+. The rate of hydrolysis of a mismatched dNMP at the primer terminus is greater in the presence of Mg2+ vs. Mn2+, whereas the rate of hydrolysis of a properly base-paired terminal nucleotide is greater in Mn2+ vs. Mg2+. These studies demonstrate that both the accuracy of base selection by the polymerase activity and the specificity of hydrolysis by the 3',5' exonuclease activity are altered by the substitution of Mn2+ for Mg2+.
Insights
DNA polymerase achieves high accuracy using kinetic differences for nucleotide selection. Replacing magnesium with manganese ions increases errors by altering polymerase insertion and exonuclease proofreading activities.
Area of Science:
- Molecular Biology
- Enzymology
- Biochemistry
Background:
- DNA polymerases are crucial for DNA replication and repair.
- Accurate nucleotide selection is vital for maintaining genomic integrity.
- The proofreading activity of DNA polymerases corrects errors during replication.
Purpose of the Study:
- To investigate the kinetic mechanisms underlying DNA polymerase I's nucleotide discrimination.
- To elucidate the role of metal ions (Mg2+ vs. Mn2+) in DNA polymerase I fidelity.
- To understand how metal ion substitution affects both polymerase insertion and exonuclease proofreading activities.
Main Methods:
- Enzyme kinetics studies using Escherichia coli DNA polymerase I (large fragment).
- Determination of apparent kinetic constants (Km and Vmax) for nucleotide insertion.
- Analysis of 3',5' exonuclease activity in the presence of Mg2+ and Mn2+.
Main Results:
- DNA polymerase I utilizes differences in Km and Vmax to distinguish between complementary and noncomplementary nucleotides.
- Substitution of Mn2+ for Mg2+ significantly increases the error rate of nucleotide insertion.
- Mn2+ reduces the catalytic rate (kcat) for correct nucleotide insertion while increasing it for mismatched nucleotides.
- Mn2+ alters the 3',5' exonuclease activity, decreasing the hydrolysis of mismatched nucleotides and increasing the hydrolysis of correctly matched nucleotides.
Conclusions:
- DNA polymerase I achieves high fidelity through precise kinetic discrimination of nucleotide substrates.
- The choice of divalent metal ion (Mg2+ vs. Mn2+) profoundly impacts DNA polymerase fidelity.
- Mn2+ compromises both the polymerase's base selection accuracy and the exonuclease's proofreading efficiency, leading to increased DNA replication errors.