Pre-Steady-State Kinetic Studies of the Fidelity of Human DNA Polymerase í†

ABSTRACT: DNA polymerase í (Polí), an X-family DNA polymerase, is preferentially expressed in secondary lymphoid tissues with yet unknown physiological functions. In this study, Polí was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme had a lifetime of <20 min at 37 °C, but was stable for over 3 h at 25 °C in an optimized reaction buffer. The fidelity of human Polí was thus determined using pre-steady-state kinetic analysis of the incorporation of single nucleotides into undamaged DNA 21/41-mer substrates at 25 °C. Single-turnover saturation kinetics for all 16 possible deoxynucleotide (dNTP) incorporations and for four matched ribonucleotide (rNTP) incorporations were measured under conditions where Polí was in molar excess over DNA. The polymerization rate (kp), binding affinity (Kd), and substrate specificity (kp/Kd) are 0.006-0.076 s-1, 0.35-1.8 íM, and (8-64) 10-3 íM-1 s-1, respectively, for matched incoming dNTPs, (2-30) 10-5 s-1, 7.3-135 íM, and (4-61) 10-7 íM-1 s-1, respectively, for mismatched incoming dNTPs, and (2-73) 10-4 s-1, 45-302 íM, and (7-1300) 10-7 íM-1 s-1, respectively, for matched incoming rNTPs. The overall fidelity of Polí was estimated to be in the range of 10-3-10-5 for both dNTP and rNTP incorporations and was sequence-independent. The sugar selectivity, defined as the substrate specificity ratio of a matched dNTP versus a matched rNTP, was measured to be in the range of 492-10959. In addition to a slow an