Kinetic Analysis of a Suicidal DNA‐crosslinking Uracil DNA Glycosylase in UDG Superfamily

Abstract

Uracil DNA glycosylase (UDG) is an enzyme to initiate base excision repair (BER) of DNA by removing the damaged base from the strand and forming an apurinic/apyrimidinic site (AP site). There are at least six families of enzymes found in UDG superfamily. All the UDGs contain three motifs in their catalytic centers, and the sequences of the motifs are conserved, guaranteeing the glycosylase activity. UDGX was a newly discovered protein of the UDG superfamily from Mycobacterium smegmatis, which was confirmed to be closer to the Family 4 UDGs (UDGa) than the other members by studying its structure and sequence. However, different with the Family 4 UDGs, an obvious loop was inserted in the motif 3 of UDGX. In addition, UDGX was the first reported UDG which can covalently crosslink to the AP site formed by base excision via its His109 on the inserted loop. Several mutants were generated by substituting the key residues in the motif 1 and motif 3 of the MsmUDGX. Uracil DNA glycosylase activity assays as well as uracil DNA crosslinking activity assays were performed with these mutants. It was observed that the E52A and D56A mutants retained both the uracil DNA glycosylase and DNA crosslinking activities, however the H109A, H109N and H109Q only retained the uracil DNA glycosylase activity but lost the DNA crosslinking activity due to the substitution of the His109. Moreover, the loss of both activities of the D59A and R107A mutants were observed. Followed by the activity assays, kinetic analyses were performed on the mutants and the UDGX wild type. The k2/Km of the wild type MsmUDGX on the G/U substrate was determined to be 1.8 ± 0.2 × 104 s‐1M‐1, which was 10‐fold lower than the k2/Km of the Family 4 UDG (TthUDGa) reported. Compared to the wild type, E52A, H109A, H109N and H109Q mutants exhibited a significant decrease of the catalytic reaction rate, suggesting that not only the key residue Glu52 in the motif 1 but His109 may also play an important role in the uracil excision process. The Asp56 and Asp59 in the motif 1 were confirmed to form salt bridges with the Arg107 on the inserted loop in the motif 3, which were hypothesized to stabilize the loop during the interaction between the enzyme and the DNA substrates. Consistent with the hypothesis, the mutations on Asp59 and Arg107 caused the loss of both activities, but the D56A mutant retained both activities, and the kinetic analysis indicated only slight decrease of the reaction rate, which may suggest the Asp56 was not the key residue to stabilize the loop in the salt bridges. In conclusion, the kinetic analysis provides a quantitative assessment on the roles of key catalytic residues on uracil excision activity and DNA‐crosslinking activity.