Summary DNA polymerase (pol) β is a model polymerase involved in gap-filling DNA synthesis utilizing two metals to facilitate nucleotidyl transfer. Previous structural studies have trapped catalytic intermediates by utilizing substrate analogues (dideoxy-terminated primer or non-hydrolysable incoming nucleotide). To identify additional intermediates during catalysis, we now employ natural substrates (correct and incorrect nucleotides) and follow product formation in real time with fifteen different crystal structures. We are able to observe molecular adjustments at the active site that hasten correct nucleotide insertion and deter incorrect insertion not appreciated previously. A third metal binding site is transiently formed during correct, but not incorrect, nucleotide insertion. Additionally, long incubations indicate that pyrophosphate more easily dissociates after incorrect, compared to correct, nucleotide insertion. This appears to be coupled to subdomain repositioning that is required for catalytic activation/deactivation. The structures provide insights into a fundamental chemical reaction that impacts polymerase fidelity and genome stability.

The molecular details of the nucleotidyl transferase reaction have remained speculative, as strategies to trap catalytic intermediates for structure determination utilize substrates lacking the primer terminus 3'-OH and catalytic Mg 2+ resulting in an incomplete and distorted active site geometry. Since the geometric arrangement of these essential atoms will impact chemistry, structural insight into fidelity strategies has been hampered. Here we present the first crystal structure of a pre-catalytic complex of a DNA polymerase 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 with two new structures of DNA polymerase β lacking the 3'-OH or catalytic Mg 2+ is described. These structures provide direct evidence that both atoms are required to achieve a proper geometry necessary for an in-line nucleophilic attack of O3' on the αP of the incoming nucleotide.

DNA apurinic-apyrimidinic (AP) sites are prevalent non-coding threats to genomic stability and are processed by AP endonuclease 1 (APE1). APE1 incises the AP-site phosphodiester backbone, generating a DNA repair intermediate that is potentially cytotoxic. The molecular events of the incision reaction remain elusive due in part to limited structural information. We report multiple high-resolution human APE1:DNA structures that divulge novel features of the APE1 reaction, including the metal binding site, nucleophile, and arginine clamps that mediate product release. We also report APE1:DNA structures with a T:G mismatch 5′ to the AP-site, representing a clustered lesion occurring in methylated CpG dinucleotides. These reveal that APE1 molds the T:G mismatch into a unique Watson-Crick like geometry that distorts the active site reducing incision. These snapshots provide mechanistic clarity for APE1, while affording a rational framework to manipulate biological responses to DNA damage.