Title

Dna Polymerase I: A Conformationally Active Enzyme (manganese Mutagenesis)

Date of Award

1987

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

Abstract

Studies on the mechanism by which DNA polymerase I of Escherichia coli discriminates between nucleotide substrates which are complementary or noncomplementary to the template have been carried out. The results suggest that DNA polymerase I is a conformationally active enzyme and that substrate-induced conformational changes result in error discrimination during DNA synthesis. Four lines of evidence support this hypothesis.1. The mechanism of base selection by the large fragment of DNA polymerase I has been investigated by determining the apparent kinetic constants for phosphodiester bond formation with dNTP substrates that are complementary and noncomplementary to the hook polymer template-primers poly(dC)-oligo(dG) and poly(dT)-oligo(dA).2. The mutagenic metal ion Mn$\sp{2+}$ lowers the specificity of base selection. The substitution of Mn$\sp{2+}$ for Mg$\sp{2+}$ as divalent metal activator does not significantly affect apparent K$\sb{\rm M}$ values for either complementary or noncomplementary nucleotides, but V$\sb{\rm max}$ values are significantly increased with noncomplementary nucleotide substrates, suggesting that effects of Mn$\sp{2+}$ on the specificity of base selection are unlikely to be mediated entirely through its interaction with dNTP substrates. Mn$\sp{2+}$ also lowers the specificity of proofreading by the 3$\sp\prime$-5$\sp\prime$ exonuclease activity of DNA polymerase I by increasing the rate of hydrolysis of complementary nucleotides and decreasing the rate of hydrolysis of mismatched nucleotides at the primer terminus. Since Mn$\sp{2+}$ alters the specificity of both base selection and proofreading, its effects are likely due to its interaction with the enzyme-DNA complex, possibly by altering the conformation of the active sites of both polymerase and exonuclease activities.3. Substitution of Mn$\sp{2+}$ for Mg$\sp{2+}$ results in a large reduction (greater than 2000-fold) in the apparent Ki values for the competitive inhibition of DNA synthesis by complementary 2$\sp\prime$,3$\sp\prime$-dideoxy-NTP analogues. Little or no effect on apparent Ki or K$\sb{\rm M}$ values is observed with dNTP, NTP, araNTP or 3$\sp\prime$-dNTP.4. Studies on the role of the primer terminus in base selection demonstrate inhibition of DNA synthesis by dideoxy-terminated DNA only when the terminal ddNMP is complementary to the template base. The formation of a dead-end complex, incapable of chain extension, is enhanced by the presence of complementary dNTP. (Abstract shortened with permission of author.)

Keywords

Chemistry, Biochemistry

Link to Full Text

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