| Popis: |
DNA polymerase ε (Polε) performs bulk synthesis of DNA on the leading strand during genome replication. Polε 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 Polε 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 Polε (hPolε), performing binding and kinetic studies at near-physiological salt concentration. Pre-steady-state kinetic studies revealed that hPolεCD extends RNA primers with approximately 3,300-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 hPolεCD has low affinity to DNA (KD of 1.5 µM). Strikingly, a change of salt concentration from 0.1 M to 0.15 M reduces stability of the hPolεCD/DNA complex by 25-fold. The incoming dNTP does not affect affinity to DNA but makes hPolε discriminative against RNA and chimeric RNA-DNA primers. In summary, our studies revealed that hPolε discrimination against RNA-containing primers is based on three main factors: incoming dNTP, 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. |
