br Concluding Remarks br Outstanding Questions br Acknowledg

Concluding Remarks
Outstanding Questions
Acknowledgments Research on DNA glycosylases in our laboratory is supported by the National Science Foundation (MCB-1517695). A.A.R. and N.P.B. are supported by the National Science Foundation Graduate Research Fellowship Program (DGE-1445197).
Introduction Cellular DNA is exposed to a wide variety of both exogenous and endogenous damaging agents, and the resulting lesions may carry mutagenic, apoptotic, or transformative potential for the cell. Nucleobase lesions, for example the deamination of C to U, are repaired via the lxr receptor excision repair (BER) pathway [1,2]. A DNA glycosylase, such as a uracil DNA glycosylase, recognizes and cleaves the glycosidic bond of the U, creating an abasic site that is subsequently incised by apurinic/apyrimidinic endonuclease 1 (APE1). Polymerase β (polβ) then removes the deoxyribosephosphate (dRP) and inserts a C opposite the unpaired G, and ligase seals the nick [1]. Humans have a total of four glycosylases responsible for removing U lesions: uracil DNA glycosylase (UNG/UDG), single-strand selective monofunctional uracil DNA glycosylase (SMUG1), thymine DNA glycosylase (TDG), and methyl binding domain 4 (MBD4) [2]. With the exception of MBD4, the human uracil glycosylases belong to the structural UDG superfamily of glycosylases [2,3]. This structural superfamily is defined by a common central α/β fold (highlighted in royal blue in Fig. 1A–C) [3]. Though all three members of the UDG superfamily remove U, their substrate specificities and roles within the cell differ dramatically. UDG has the narrowest range of substrates and is able to excise U from single-stranded (ss) DNA, U opposite A or G from duplex DNA, and 5-fluorouracil (5FU) opposite A [[4], [5], [6]]. UDG is expressed at high levels and is upregulated during S-phase to remove U misincorporated from the dNTP pool [6]. It is noteworthy that UDG is the only member of the superfamily to have a mitochondrial isoform (UNG1), which alone is responsible for the removal of U from the mitochondrial genome [3,7]. SMUG1 has increased substrate tolerance relative to UDG, with the ability to excise U from ssDNA, U opposite A or G, and halogenated and oxidized uracil derivatives [6,[8], [9], [10], [11]]. Unlike the other two members of the superfamily, SMUG1 does not undergo cell cycle regulation, but rather is expressed at constant, low levels [6]. Interestingly, SMUG1 tends to accumulate in nucleoli [6], which contain both regions of active transcription and condensed chromatin [12]. Like SMUG1, TDG has a wider substrate range than UDG, able to excise U or T opposite G, and a variety of U and C derivatives, including the oxidation products of the epigenetic silencer 5-methylcytosine (5mC), from duplex DNA with greater efficiency in CpG sequence contexts [[13], [14], [15], [16], [17], [18], [19], [20], [21], [22]]. TDG, furthermore, interacts with a diverse array of proteins, including chromatin remodelers, transcription factors, and other DNA repair proteins [[23], [24], [25], [26], [27], [28], [29]]. The regulation of TDG is maintained through post-translational modifications (PTM) [23,28,[30], [31], [32], [33]] and degradation of the protein at the G1/S transition [34,35]. The biochemical activities of these glycosylases have been characterized in duplex DNA, however there have been fewer examinations of UDG and SMUG1 activity on chromatin or packaged DNA [[36], [37], [38], [39], [40]]. The studies on packaged DNA have revealed that UDG and SMUG1 activity is generally lower than on duplex, and that local dynamics of the DNA and associated proteins can modulate activity. There are not any reports of TDG activity on packaged DNA. The nucleosome core particle (NCP) is the most basic unit of DNA packaging [41]. It consists of 145–147 bp wrapped ∼1.7 times around a core of histone proteins and has a two-fold rotational axis of pseudosymmetry called the dyad axis (Fig. 1D). We recently reported that UDG is unique among a group of glycosylases representing four structural superfamilies in its ability to excise an outward-facing and highly solution-accessible lesion at the often-studied dyad axis region of an NCP with activity comparable to duplex [38]. These results led us to question whether the high activity is a property of the entire UDG superfamily or solely of UDG. Here we report that, unlike UDG, SMUG1 exhibits sharply reduced activity for excising an outward-facing U at the dyad relative to U in duplex DNA, while TDG displays intermediate levels of activity. Implications of these observations for the potential biological roles of these glycosylases is discussed.