Crystallographic evidence for two‐metal‐ion catalysis in human pol η

Extensive evidence exists that DNA polymerases use two metal ions to catalyze the phosphoryl transfer reaction. Recently, competing evidence emerged, suggesting that a third metal ion, known as MnC, may be involved in catalysis. The binding of MnC was observed in crystal structures of the replication complexes of human polymerase (pol) η, pol β, and pol μ. Its occupancy ( q MnC ) in the pol η replication complexes exhibited a strong correlation with the occupancy of the formed product pyrophosphate ( q PPi ), i.e., q MnC ∝ q PPi . However, a key piece of information was missing that is needed to distinguish between two possible sequences of events: (i) the chemical reaction occurs first with only two meal ions, followed by the binding of MnC in a “catch‐the‐product” mode; and (ii) MnC binds first, followed by the chemical reaction with all three metal ions in a “push‐the‐reaction‐forward” mode. Both mechanisms can lead to a strong correlation between q MnC and q PPi . However, q MnC ≤ q PPi in the first scenario, whereas q MnC ≥ q PPi in the second. In this study, an analysis of crystallographic data published recently for pol η complexes shows that the formation of the product pyrophosphate definitely precedes the binding of MnC. Therefore, just like all other DNA polymerases, human pol η employs a two‐metal‐ion catalytic mechanism. Rather than help to catalyze the reaction, MnC stabilizes the formed product, which remains trapped inside the crystals, before it slowly diffuses out.