The DNA cleavage activity assay in the present study

The DNA cleavage activity assay in the present study also demonstrated the DNA glycosylase activities of SMUG1, NEIL1, TDG, and NTHL1 for 5OHU paired with A. While our findings on the activities of SMUG1 and NEIL1 were consistent with the results reported from previous studies [25], [50], the activities of TDG and NTHL1 for 5OHU:A were novel findings. Since no DNA glycosylase activity for A paired with 5OHU was detected in our analysis, 5OHU:A appears to lead to 5OHU excision and formation of a T:A base-pair (i.e., induction of C→T mutation); however, even if 5OHU is not excised, 5OHU:A may result in a C→T mutation or the presence of residual 5OHU as a consequence of the next round of DNA replication, as shown in Suppl. Fig. S3C. Thus, DNA glycosylase activity for 5OHU:A would not greatly affect the induction of C→T mutation. This is consistent with our results which suggested that not only overexpression of DNA glycosylase proteins possessing glycosylase activity for 5OHU:G, but also that of proteins possessing glycosylase activity for both 5OHU:G and 5OHU:A in human WZB117 sale leads to the suppression of 5OHU-induced mutations.
Introduction Formamidopyrimidine–DNA glycosylase (Fpg, also known as MutM) is a bacterial repair enzyme that excises pre-mutagenic 8-oxoguanine (oxoG) and a number of other oxidatively damaged bases from DNA. Fpg together with its bacterial paralog endonuclease VIII (Nei) define a structural Fpg/Nei superfamily, which also includes plant MutM homologs (MMH proteins) and endonuclease VIII-like (NEIL) proteins found in vertebrates and some giant viruses. Structurally, they all share a two-domain organization, with an N-terminal β-sandwich domain equipped with a catalytic α-helix and a C-terminal domain bearing a DNA-binding helix–two-turn–helix (H2TH) motif and either a single β-hairpin zinc finger or a zincless finger motif [1,2]. Despite this similar general organization, the active sites of these enzymes are sculpted in different ways, allowing them to recognize and excise a wide array of damaged bases. A number of X-ray structures for Fpg from Escherichia coli, Geobacillus stearothermophilus, Lactococcus lactis, and Thermus thermophilus have been reported [[3], [4], [5], [6]]. Understanding of the reaction mechanism of Fpg greatly benefited from the series of structures of G. stearothermophilus Fpg sampling the reaction path from initial lesion encounter to the final pre-catalytic complex [5,[7], [8], [9], [10], [11], [12], [13], [14]] enhanced by molecular dynamic (MD) and QM/MM simulations [[9], [10], [11], [12],[15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]] and pre-steady state enzyme kinetics [23,[26], [27], [28], [29]]. Thus, the mechanistic functions of many catalytically important residues in Fpg are currently understood with the atomic spatial resolution and millisecond time resolution. It has been established, for example, that the residue Phe110 (E. coli numeration) is inserted into undamaged DNA early in the reaction and buckles the sampled base pair, which opens if it contains oxoG instead of G, that Arg108 distorts undamaged DNA allowing the enzyme to walk between adjacent base pairs, and that after the base pair opening Arg108 and Met73 are inserted into the base stack instead of oxoG thereby locking oxoG in a catalytically accessible conformation in the enzyme’s active site. This combination of structural studies with kinetics and modeling is powerful but tends to provide information on a narrow set of residues that, as made obvious by the structure, reside at or near the active site. At the same time, long-range effects that propagate through the protein globule are well known in enzyme catalysis [[30], [31], [32]]. Functionally important residues that lie far away from the active site are usually highly conserved, and some of these residues have been predicted in Fpg and other Fpg/Nei enzymes by conservation analysis and shown to be required for efficient catalysis and/or substrate specificity [[33], [34], [35], [36]]. Moreover, it is also well known that residue pairs involved in functionally important interactions within the protein globule tend to coevolve, resisting changes that disrupt their interaction [[37], [38], [39], [40]]. For example, to maintain critical salt bridges after one member of the pair reverses the charge, the selection would favor a second mutation with exactly the opposite reversal at the other position of the bridge. In this work, we have analyzed residue coevolution in the Fpg proteins to search for intramolecular interactions important for the function of this repair enzyme.