br Conclusion br Conflict of interest statement br

Conclusion
Conflict of interest statement
Acknowledgements This work was supported by the National Science Foundation (MCB-1817417 to S.D.) and National Institutes of Health (R01-GM72711 to A.C.D.). We thank members of the Delaney laboratory for careful reading of the manuscript and helpful discussion.
Introduction Reactive oxygen species appear in cells as byproducts of aerobic respiration and may be also generated by environmental sources such as various chemicals, UV and ionizing radiation [1], [2]. They react with DNA to produce a variety of genotoxic lesions that contribute to aging and a number of diseases [2], [3], [4]. Oxidized DNA moieties are recognized and removed by the Dynasore excision repair (BER) system, which includes lesion-specific DNA glycosylases, apurinic/apyrimidinic (AP) endonucleases, DNA polymerases and DNA ligases [5], [6]. DNA glycosylases are responsible for finding chemically modified bases and catalyzing their excision from DNA. Two Escherichia coli DNA glycosylases belonging to the helix–two-turn–helix (H2TH) superfamily, formamidopyrimidine–DNA glycosylase (Fpg) and endonuclease VIII (Nei), remove oxidized purines and pyrimidines, respectively [7], [8]. In human cells, three Nei-like proteins, NEIL1, NEIL2, and NEIL3, have been characterized [8], [9]. NEIL1 has broad substrate specificity, removing both pyrimidine- and purine-derived lesions from DNA [10], [11], [12], [13], [14], [15]. NEIL2 activity primarily excises oxidative derivatives of cytosine and has preference for the lesion in bubbles resembling transcription intermediates [16], [17], [18]. NEIL3 mainly removes advanced products of purine oxidation from single-stranded DNA and has a very restricted tissue distribution suggestive of a role in development or cell regulation [19], [20]. Members of the H2TH superfamily are bifunctional enzymes capable of catalyzing hydrolysis of the N-glycosidic bond followed by β,δ-elimination, thus introducing a single-nucleoside gap into DNA [7], [21]. They all use an N-terminal amino group (Val1 in NEIL3, Pro1 in all other homologs) to attack at C1′ of the damaged nucleotide and form a Schiff base-type covalent reaction intermediate [16], [22], [23], [24]. Several three-dimensional structures of Fpg, Nei, and NEILs as well as their complexes with DNA have been determined [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38]. The recognition of the substrate lesions by these enzymes involves extensive conformational changes in both protein and DNA, such as closing of the protein domains, DNA kinking, damaged base eversion out of the DNA helix into the enzyme's active site and intrusion of several amino acid side chains into DNA. In particular, H2TH proteins diverge in two important structural elements: the residues that are inserted into the void created in DNA by the eversion of the damaged nucleotide, and the “missing loop” that forms the base-recognition pocket and is disordered in all DNA-bound structures lacking the cognate base. Previously, we had applied stopped-flow kinetics with fluorescence detection to analyze the dynamics of conformational transitions during substrate binding and cleavage by two members of the H2TH superfamily, E. coli Fpg and Nei [39], [40], [41], [42], [43], [44], [45], [46], [47], [48]. Based on several detectable sequential changes in the Trp fluorescence of Fpg, using a series of DNA substrates of different structure, and supplementing this with other reporters (2-aminopurine, pyrrolocytosine, 1,3-diaza-2-oxophenoxazine, 3-hydroxychromone) strategically incorporated at different places in DNA, we have been able to single out and attribute five reversible steps in the lesion recognition followed by an irreversible step, presumably corresponding to the reaction chemistry, and then the product release step (Scheme 1). In Fpg, the sequential reversible steps in Scheme 1 correspond to the following: (i) non-specific primary encounter, (ii) initial recognition with the destabilization of the DNA around the lesion with the insertion of Phe110, (iii) kinking the DNA axis, (iv) eversion of oxoG base from the double helix into the enzyme's active site and filling the resulting void in the double helix by Arg-108 and Met-73, and (v) isomerization of the enzyme to a catalytically competent conformation. With Nei, only three reversible steps were observed by Trp fluorescence before the irreversible one, and the second of them corresponded to two DNA conformational changes revealed by 3-hydroxychromone reporter fluorescence. The similarity of Fpg and Nei structures suggests that their substrate recognition mechanisms should be similar, yet some of the steps may remain not discernible if Trp residues are located unfavorably in the protein structure.