Human DNA Polymerase {nu} Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency [Enzymology]

May 11th, 2015 by Gowda, A. S. P., Moldovan, G.-L., Spratt, T. E.

DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand crosslink repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. Pol ν binds to DNA with a KdDNA of 9.2 nM, with an off rate constant of 0.013 s-1 and a on rate constant of 14 µM-1 s-1. dNTP binding is rapid with a Kds of 20 and 476 µM for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a KdPPi of 3.7 mM and a maximal rate constant of 11 s-1. Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with a rate constants of 75 s-1 and 20 s-1, rapid phosphodiester bond formation occurs with a Keq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 s-1` and 0.5 s-1, for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. Pol ν catalyzes both correct and mispair formation with high catalytic efficiency.