Through a large scale phylogenetic

Through a large-scale phylogenetic analysis of UDG superfamily in more than 1000 completely sequenced genomes, it is shown that family 3 Bikinin sale can be separated into two clades with the traditional family 3 SMUG1 as one of them (Fig. 1) [31]. UDG enzymes in another clade are present in eubacteria genus including listeria, lactobacillus, Streptomyces, Amycolatopsis and Flavobacteriaceae. This group of UDGs, which we named as SMUG1-like, is more similar to family 3 SMUG1s and shares a common ancestor with family 1 UNGs (Fig. 1). As identified previously [31], a distinct difference between SMUG1-like and traditional SMUG1 is that the “GMNPGP” in motif 1 of SMUG1 is changed to “GSSPAR” in SMUG1-like enzymes (Fig. 2A). To our knowledge, the biochemical and enzymatic properties of SMUG1-like enzymes are completely unknown. In this study, we characterized a SMUG1-like DNA glycosylase from Listeria innocua (Lin). To our surprise, Lin SMUG1-like glycosylase is a single-stranded UDG with little activity on double-stranded uracil-containing DNA. Mutational analysis indicates similar roles of active site residues corresponding to SMUG1. Interestingly, a double substitution of S67-S68 in Lin SMUG1-like DNA glycosylase by M67-N68 in motif 1 to mimic SMUG1 is able to partially rescue the negative effects of the S67M and S68N single mutants, which implies correlation and co-evolution of the two catalytic residues. The potential physiological role of the SMUG1-like is also discussed.
Material and methods
Results and discussion
Funding This project was supported in part by the National Institutes of Health (GM121997 to W.C.) and the Greenwood Genetic Center (to L.W.).
Conflict of interest
Acknowledgements We thank Dr. Min Cao (Clemson University) for the Listeria innocua strain and Dr. Richard P. Cunningham (State University of New York, Albany, New York) for E. coli strains. We also thank members of Cao laboratory for assistance and discussions.
Introduction Thymine DNA glycosylase (TDG) plays an extremely essential role in defense of genetic mutations, maintenance of genetic integrity and study of DNA active demethylation mechanism [1]. TDG can selectively remove the mismatched base to generate apyrimidinic (AP) site through N-glycosidic bond hydrolysis and subsequently initiates the base replacement by downstream base excision repair (BER) pathway both in vitro and in mammalian cells [[2], [3], [4], [5], [6]]. In the BER pathway, TDG can remove thymine (T) moieties of guanine: thymine (G: T) mismatched base pairs which were formed during process of 5-methylcytosine (5-mC) deamination [7,8], some other lesions can also be removed, such as uracil (U) from G: U mismatch and 5-hydroxymethyluracil (5-hmU) from G: 5hmU mismatch [9,10]. In DNA active demethylation process, TDG can abscise 5-formylcytosine and 5-carboxylcytosine which were formed via sequential oxidation of 5-mC [2,11]. Since TDG has such important biological functions, it is crucial to search for and develop a method for TDG analysis with high sensitivity, selectivity and convenience. Until now, some approaches have been reported to assess TDG activity based on different principles or mechanisms, among these methods including gel electrophoresis [[12], [13], [14], [15]], fluorescence assay [[16], [17], [18]], electrochemical method [19] etc. Although these methods present respective advantages, some limitations still exist. Time consumption in gel electrophoresis, short lifetime of fluorescence in organic fluorescent compounds, unsatisfying sensitivity for electrochemical method are the drawbacks of these methods. It is of great importance to choose the appropriate method when detecting a target. Electrogenerated chemiluminescence (ECL) is a process in which highly reactive species are applied with high voltage at electrodes interface and arousing high-energetic electron transfer reactions which transformed reactive species into excited state that emitting light [20,21]. Over the past decades, biosensors based on ECL have received increasing concern because of their high sensitivity, fast detection, easy operation and simple device. To our best knowledge, ECL biosensing methods for the detection of TDG have not been reported yet. Therefore, the exploration of a novel ultrasensitive ECL biosensing method to evaluate TDG activity is highly desirable.