Efficient discrimination against RNA-containing primers by human DNA polymerase epsilon

DNA polymerase epsilon (Pol epsilon) performs bulk synthesis of DNA on the leading strand during genome replication. Pol epsilon binds two substrates, a template:primer and dNTP, and catalyzes a covalent attachment of dNMP to the 3' end of the primer. Previous studies have shown that Pol epsilon easily inserts and extends ribonucleotides, which may promote mutagenesis and genome instability. In this work, we analyzed the mechanisms of discrimination against RNA-containing primers by human Pol epsilon (hPol epsilon), performing binding and kinetic studies at near-physiological salt concentration. Pre-steady-state kinetic studies revealed that hPol epsilon(CD) extends RNA primers with approximately 3300-fold lower efficiency in comparison to DNA, and addition of one dNMP to the 3 ' end of an RNA primer increases activity 36-fold. Likewise, addition of one rNMP to the 3 ' end of a DNA primer reduces activity 38-fold. The binding studies conducted in the presence of 0.15 M NaCl revealed that human hPol epsilon(CD) has low affinity to DNA (K-D of 1.5 mu M). Strikingly, a change of salt concentration from 0.1 M to 0.15 M reduces the stability of the hPol epsilon(CD)/DNA complex by 25-fold. Upon template:primer binding, the incoming dNTP and magnesium ions make hPol epsilon discriminative against RNA and chimeric RNA-DNA primers. In summary, our studies revealed that hPol epsilon discrimination against RNA-containing primers is based on the following factors: incoming dNTP, magnesium ions, a steric gate for the primer 2 ' OH, and the rigid template:primer binding pocket near the catalytic site. In addition, we showed the importance of conducting functional studies at near-physiological salt concentration.