Speaker
Description
Reaction intermediates during DNA synthesis have been studied in detail using time-resolved
X-ray crystallography for translesion and repair DNA polymerases. Contrary to the originally
proposed two-metal-ion mechanism, a third metal ion was identified between the finger
domain and the α- and β-phosphates of the incoming nucleotide. This third metal ion was
suggested to either participate in catalysis or stabilize product formation. To investigate this
further in a replicative polymerase, we conducted time-resolved X-ray crystallography with
DNA Polymerase epsilon, which synthesizes DNA at a much faster rate—10x to 100x higher
than family Y and X polymerases. Surprisingly, no metal ion was observed between the finger
domain and the α- and β-phosphates of the incoming nucleotide in any of the solved structures
with Pol epsilon. Instead, our biochemical and structural data support the original two-metal
mechanism. In addition, we discovered that the 3’-OH group releases a proton, which is
channeled via structural waters to a basic residue in the Palm domain. After forming a new
bond with the incoming nucleotide, an acidic residue in the finger domain protonates the
released pyrophosphate, stabilizing the product. In summary, it seems that metal A’s role is
to lower the pKa of the 3’-OH group, followed by specific residues in Pol epsilon donating or
receiving a proton to catalyze this acid-base reaction.
Co-author: Erik Johansson
