Pre-steady-state Kinetic Characterization of the DinB Homologue DNA Polymerase of Sulfolobus solfataricus

Janina Cramer,Tobias Restle

doi:10.1074/jbc.m504481200

Abstract

Equilibrium as well as pre-steady-state measurements were performed to characterize the molecular basis of DNA binding and nucleotide incorporation by the thermostable archaeal DinB homologue (Dbh) DNA polymerase of Sulfolobus solfataricus. Equilibrium titrations show a DNA binding affinity of about 60 nm, which is approximately 10-fold lower compared with other DNA polymerases. Investigations of the binding kinetics applying stopped-flow and pressure jump techniques confirm this weak binding affinity. Furthermore, these measurements suggest that the DNA binding occurs in a single step, diffusion-controlled manner. Single-turnover, single dNTP incorporation studies reveal maximal pre-steady-state burst rates of 0.64, 2.5, 3.7, and 5.6 s(-1) for dTTP, dATP, dGTP, and dCTP (at 25 degrees C), which is 10-100-fold slower than the corresponding rates of classical DNA polymerases. Another unique feature of the Dbh is the very low nucleotide binding affinity (K(d) approximately 600 mum), which again is 10-20-fold lower compared with classical DNA polymerases as well as other Y-family polymerases. Surprisingly, the rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. Thus, unlike replicative polymerases, an "induced fit" mechanism to select and incorporate nucleotides during DNA polymerization could not be detected for Dbh.

Highlights

Polymerases involved in genome replication accomplish their task with high fidelity, ensuring genome stability
The x-ray structures of three nonhuman Y-family DNA polymerase members, the N-terminal catalytic domain of yeast DNA polymerase ␩ and Solfolobus solfataricus (P1 and P2, respectively) DNA polymerases Dbh (DinB homologue) and Dpo4 (DNA polymerase IV), the latter crystallized in a ternary complex with DNA and an incoming nucleotide, reveal that these proteins have a high degree of structural homology [11,12,13,14]
We provide experimental evidence that nucleotide insertion and misinsertion by Dbh do not follow an induced “fit mechanism,” indicating a polymerase pathway different from that observed with classical DNA polymerases as well as with other Y-family polymerases

Summary

Introduction

Polymerases involved in genome replication accomplish their task with high fidelity, ensuring genome stability. We report on steady-state and pre-steady-state kinetic analyses, aiming to unravel mechanistic details of DNA binding and nucleotide incorporation by the Y-family Dbh polymerase. Performing an equivalent incorporation experiment as described above under multiple-turnover conditions (excess of p/t over enzyme; 1.5 ␮M and 100 nM, respectively), we observe a burst of product formation followed by a slower steady-state phase with a rate of about 0.2 sϪ1 (data not shown).

Results

Conclusion