Kinetic Mechanism of the 3‘ → 5‘ Proofreading Exonuclease of DNA Polymerase III. Analysis by Steady State and Pre-Steady State Methods

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Abstract
DNA polymerase III holoenzyme is the major replicative enzyme in Escherichiacoli. An important component of the high-fidelity DNA synthesis that is characteristic of DNA polymerase III holoenzyme is the 3‘ → 5‘ proofreading exonuclease activity resident in the ε subunit. Steady state and pre-steady state conditions have been used to determine equilibrium and Michaelis constants for substrate binding and the rate constant for cleavage by purified ε subunit. The steady state kinetic constants are Km = 16 ± 6 μM and kcat = 210 ± 23 s-1 for degradation of single-stranded DNA by ε. These steady state values are in agreement with the rate constants determined for excision of the 3‘ nucleotide of a dT10 oligomer under pre-steady state conditions. Using a simple two-step model, E + Dn ⇌ E·Dn → E + Dn-1, we find K = 12 μM and kf = 280 s-1 for the dT10 substrate. In these experiments, ε subunit acts in a distributive manner and product release is not the rate-limiting step. Activity of the ε subunit on paired DNA oligonucleotides with zero to three mismatches at the 3‘ terminus indicates that an additional step is required in the mechanism. In the scheme Dn ⇌ Dn* + E ⇌ E·Dn* → E + Dn-1, the 3‘ terminus undergoes a conformational change or “melts” before the DNA is a substrate for ε subunit. With this additional step, the values for binding of activated substrate and cleavage are the same as those for single-stranded DNA. The kinetics for exonucleolytic degradation of single-stranded, paired, and mispaired oligonucleotides support the model that the rate-limiting step in exonucleolytic proofreading of DNA by ε subunit is the DNA-melting step.

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