Dissertations from ProQuest

Title

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

1987

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