Kinetic Analysis of Correct Nucleotide Insertion by a Y-family DNA Polymerase Reveals Conformational Changes Both Prior to and following Phosphodiester Bond Formation as Detected by Tryptophan Fluorescence

Jeff W. Beckman,Qixin Wang,F. Peter Guengerich

doi:10.1074/jbc.m806785200

Jeff W. Beckman, Qixin Wang + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m806785200

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Abstract

The Sulfolobus solfataricus Y-family DNA polymerase Dpo4 is a model for translesion replication and has been used in the analysis of individual steps involved in catalysis. The role of conformational changes has not been clear. Introduction of Trp residues into the Trp-devoid wild-type protein provided fluorescence probes of these events, particularly in the case of mutants T239W and N188W. With both mutants, a rapid increase in Trp fluorescence was observed only in the case of normal base pairing (G:C), was saturable with respect to dCTP concentration, and occurred in the absence of phosphodiester bond formation. A subsequent decrease in the Trp fluorescence occurred when phosphodiester bond formation was permitted, and these rates were independent of the dCTP concentration. This step is relatively slow and is attributed to a conformational relaxation step occurring after pyrophosphate release, which was measured and shown to be fast in a separate experiment. The measured rate of release of DNA from Dpo4 was rapid and is not rate-limiting. Overall, the measurements provide a kinetic scheme for Dpo4 different than generally accepted for replicative polymerases or proposed for Dpo4 and other Y-family polymerases: the initial enzyme.DNA.dNTP complex undergoes a rapid (18 s(-1)), reversible (21 s(-1)) conformational change, followed by relatively rapid phosphodiester bond formation (11 s(-1)) and then fast release of pyrophosphate, followed by a rate-limiting relaxation of the active conformation (2 s(-1)) and then rapid DNA release, yielding an overall steady-state kcat of <1 s(-1).

Highlights

Whether these two major enzyme classes catalyze polymerization is not yet clear
It is believed that replicative polymerases bind DNA tightly, followed by binding a correct dNTP that instigates an “induced-fit” conformational change to form an active ternary complex leading to high efficiency and fidelity polymerization, e.g. [7]
The model preunmodified DNA by Dpo4, rate dicts a complete conversion of substrate DNAG to product at constants were added to a classic minimal model [42], with an each dCTP concentration, but the possibility exists that an adjustment to have a conformational change occur after the additional, unproductive step within the catalytic cycle negrelease of PPi (Fig. 9A)

Summary

EXPERIMENTAL PROCEDURES

Materials—All reagents were of the highest quality commercially available. Unlabeled dNTPs were purchased from New England Biolabs (Beverly, MA). Excess wild-type or mutant Dpo (1–5 ␮M), incubated with 300 nM primer-labeled DNAG, was rapidly mixed with 5 mM MgCl2 and various concentrations of dNTP to obtain the nucleoside triphosphate-dependent rate of polymerization, kp. A mixture of 500 ␮M dCTP, 5 mM MgCl2, and 1.1 ␮M PBP-MDCC from one syringe was rapidly mixed with a mixture of 1.2 ␮M Dpo T239W and 200 nM DNAG from the other, and the resulting Pi binding to PBP-MDCC (following release of PPi due to DNA polymerization and rapid hydrolysis of PPi to Pi by PPase) was observed by monitoring the increase in fluorescence emission at 465 nm For analysis of ternary complex DNAGdd off-rates, Dpo mutant Y12W was added in 5-fold excess to a 1 ␮M

RESULTS

Fluorescence emission spectra were recorded for these

Findings

DISCUSSION