Fig. 3. Characterization of the reaction of 3′-EUMP catalyzed by reconstituted PolH. (A) HPLC analysis of the activity assay (monitoring at 260 nm). (1) PolH with 3′-EUMP, SAM and dithionite; (2) without SAM; (3) with denatured PolH; (4) 3′-EUMP standard; (5) 5′-dA standard; (6) SAM standard; (7) purified PolH reaction product (OAP); (8) co-injection of OAP with 3′-EUMP; (B) ESI-MS/MS (78.957, 96.967, 111.018, 295.056, 350.028, and 393.034 (from left to right)); (C) ¹H-NMR of the purified product (OAP) from the PolH reaction; (D) the reaction scheme of PolH. *The impurities from background reactions in the HPLC assay or contamination from glycerol in the ¹H-NMR sample.