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  • DL-AP5 TDG furthermore is inhibited in

    2021-10-14

    TDG, furthermore, is inhibited in its excision of U on the NCP relative to duplex, though not to the same extent as SMUG1. There is no significant difference between TDGFL and TDG82−308, consistent with a previous report that the N- and C-termini missing from TDG82−308 do not affect the excision of U or T from duplex [19]. Nevertheless, there is a proportion of the NCP substrate inaccessible to glycosidic bond cleavage by TDG. This inaccessible substrate may be a population of TDG-NCP complexes for which U extrusion and/or intercalation of R275 is inhibited. On the other hand, this proportion of the substrate may be inaccessible to initial binding of TDG, due to steric clash of the enzyme with the histone core. Excision of T from a T:G wobble bp in the dyad region is significantly inhibited, with product yields less than 10% for both TDGFL and TDG82−308. Due to the increased steric hindrance of extruding T versus U, T is excised at a slower rate than U [61]. Furthermore, it is known that a specific conformation of the N-terminus is required for the recognition and removal of T and deletion of the entire N-terminus results in loss of the ability to excise T [15,62]. Indeed, it has been demonstrated that more stable binding of TDG to the DNA is required for the excision of T versus U [63]. Steric clash of the enzyme with the histone core may prevent productive binding to the NCP, thus resulting in low product yields. Since TDG is degraded prior to S-phase and cannot take advantage of the more duplex-like character of DNA during this phase of the DL-AP5 [34,35], the question remains as to which factors, if any, may increase the efficiency of removal of U and T. Possible mechanisms may include PTM of TDG or the histone proteins, interaction with and/or the presence of chromatin remodelers, and interactions with other proteins. TDG has been demonstrated to be acetylated [23,28,29,32] and phosphorylated [32] on its N-terminus, and SUMOylated [30,31,33,64,65] on its C-terminus. Phosphorylation has been demonstrated to increase activity, SUMOylation severely inhibits excision while augmenting APE1-induced product release, and acetylation has either stimulatory or inhibitory effects, based on the lesion in question [28,32,33,65]. TDG also interacts with a wide variety of chromatin remodelers, including the methyltransferases DMT3A [26], DMT3B [27], the lysine acetyltransferase CBP/p300 [23,29], and the NAD+-dependent deacetylase SIRT1 [28]. TDG has been demonstrated to interact with several other proteins, including the nucleotide excision repair protein XPC [24] and the transcription factor estrogen receptor α [66]. The presence and/or absence of these factors separately or in combination may modulate the efficiency of TDG, resulting in precise spatiotemporal activity. In light of our results, it is curious that UDG is unique among the UDG superfamily glycosylases in its rapid and complete excision of an outward-facing U at the dyad. SMUG1 and TDG, on the other hand, do not convert the substrate fully to product. It is noteworthy that UDG differs from TDG and SMUG1 in its intercalating residue, which stabilizes the duplex when the U is extruded from the helix. While the intercalating residue of UDG is a leucine [67], both SMUG1 and TDG use an arginine [9,18,19,59,60]. The positive charge on the arginine may cause electrostatic repulsion from the overall positive charge of the histone core, thus limiting the intercalation step and preventing glycosidic bond cleavage. Indeed, a mutation of the intercalating leucine in UNG to arginine (L272R) modestly decreases steady-state kinetic parameters kcat, Km, and kcat/Km on duplex substrates [68]. The helical wedge of SMUG1 presents another barrier to intercalation, as the rather constrained DNA at the dyad axis must be disrupted further for catalysis to occur. Given these results, UDG may be the main glycosylase for excision of outward-facing U near the dyad axis, with roles for SMUG1 and TDG in specific spatiotemporal and/or sequence contexts, perhaps aided by the presence of other factors and/or PTM.