Elementary steps in the DNA polymerase I reaction pathway.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• DNA polymerase I (Pol I) from Escherichia coli is crucial for DNA replication and repair.
• Understanding the kinetic mechanism of Pol I is essential for elucidating DNA synthesis processes.

Purpose of the Study:

• To investigate the polymerization mechanism of Escherichia coli DNA polymerase I (Pol I).
• To determine the kinetic parameters governing Pol I's interaction with template-primer and nucleotide incorporation.

Main Methods:

• Utilized a homopolymer template-primer system: poly(dA).oligo(dT).
• Employed isotope-partitioning experiments to elucidate reaction mechanism and kinetics.
• Analyzed rapid quench time courses to identify rate-determining steps.

Main Results:

• Pol I exhibits an ordered reaction mechanism, binding template-primer before MgTTP.
• Enzyme-template-primer binding parameters: kon = 1.2 X 10(6) M-1 s-1, koff = 0.25 s-1, KD = 2 X 10(-7) M.
• Demonstrated processive synthesis, with Pol I incorporating 40-50 nucleotides per binding event, and identified two partially rate-determining steps.

Conclusions:

• The polymerization reaction catalyzed by Pol I proceeds via an ordered mechanism with distinct substrate binding and catalytic steps.
• Processive synthesis is governed by partitioning between polymerization and enzyme dissociation.
• The initial rate-determining step likely involves a first-order isomerization preceding phosphodiester bond formation.