Strategic regulation of excision repair through structural and chemical biology

DNA polymerases employ a two‐metal (Mg 2+ ) mechanism for nucleotide insertion. These enzymes must select and incorporate a complementary deoxynucleotide from a pool of structurally similar molecules to preserve the integrity of the genome during replication and repair. The molecular details of this nucleotidyl transferase reaction have remained speculative, as strategies to trap catalytic intermediates for structure determination have utilized substrates lacking the primer terminus 3′‐OH or the catalytic Mg 2+ ; since these atoms are essential for chemistry, an understanding of geometric and electronic factors important for catalysis has been incomplete. To clarify the roles of these and other key atoms, we obtained a crystal structure of a pre‐catalytic complex of human Pol β with bound substrates that include the primer 3′‐OH and catalytic Mg 2+ . This catalytic intermediate was trapped with a non‐hydrolyzable deoxynucleotide analogue. Comparison of this new structure with those lacking the primer 3′‐OH or catalytic Mg 2+ will be described, along with computational approaches to elucidating the activation barrier to the transition state. Binding of the catalytic Mg 2+ induces conformational adjustments that result in octahedral geometry ideal for in‐line nucleophilic attack of the 3′‐oxygen on the α P of the incoming nucleotide. Progress in understanding this nucleotidyl transferase reaction will serve as a guide for development of structure‐specific small molecule probes and inhibitors for Pol β, a key base excision repair enzyme. This work was supported by the Intramural Research Program of the National Institutes of Health (NIEHS).