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    • In contrast to UDG SMUG exhibits dramatically

    2022-08-09

    In contrast to UDG, SMUG1 exhibits dramatically lower product yield in its excision of U, and this is not dependent on sequence context. Though we cannot rule out that a small amount of the product is derived from a duplex contaminant, our extensive purification techniques limit the amount of contaminating duplex in the NCP sample. The limited amount of duplex (<5%) combined with the observation that SMUG1 exhibits biphasic kinetics on this substrate lead us to conclude that some product results from SMUG1 excising U from the NCP. Due to the low product yield associated with kobs-fast, we cannot rule out that this fast phase represents SMUG1 acting on a small amount of duplex. In this case, kobs-slow represents SMUG1 acting on NCP. The product yield reveals that only ∼20% of the NCP population is accessible to SMUG1. On the other hand, both phases may reflect SMUG1 acting on the NCP. The fast phase would therefore correspond to SMUG1 excising U on a readily accessible population, while the slow phase reflects a population that requires, for example, a conformational change prior to SMUG1 binding and/or cleavage. Nevertheless, a substantial proportion of the NCP substrate is inaccessible to glycosidic bond cleavage by SMUG1. The significant inhibition of SMUG1 on the NCP substrate may be due, at least in part, to the presence of an additional α-helix directly C-terminal to its intercalation loop [9]. Though UDG, SMUG1, and TDG all possess a similar intercalation loop, SMUG1 is unique in its possession of this additional α-helix, which forms a “helical wedge” (highlighted orange in Fig. 1B) and is believed to promote DNA distortion and U extrusion [9]. In contrast to UDG and TDG, which disrupt only the plk inhibitor pair containing the scissile U, SMUG1 also disrupts the base pair on the 5′-side of the U, leading to a greater distortion of the DNA helix (9). In the context of an outward-facing lesion at the dyad, the torsional flexibility of the DNA is significantly decreased relative to duplex DNA in solution [47,54], and may provide a barrier to DNA distortion necessary for glycosidic bond cleavage. This barrier to DNA distortion may be responsible for the low product yields observed for the excision of U from NCP. In this case, the requirement for DNA distortion may also prevent efficient repair of sites that are less solution accessible. In contrast, lesions displaced from the dyad, especially at the DNA ends where there are increased dynamics [[55], [56], [57], [58]], may be more easily repaired by SMUG1. The results then bring into question whether an outward-facing U at the dyad is a substrate for SMUG1 in a cellular context. It may be the case that SMUG1 relies on chromatin remodelers, transcriptional machinery, or the degree of DNA packaging variable with the cell cycle to make lesions, both at the dyad and throughout the NCP, more accessible. Conversely, these lesions at the dyad may be removed primarily by UDG, and thus may seldom be a substrate for SMUG1. While SMUG1 may defer to UDG for lesion removal in some contexts, it tends to accumulate in nucleoli, which lack UDG [6]. Therefore, there may be factors specific to nucleoli that increase SMUG1 activity on packaged DNA. Conversely, SMUG1 may take advantage of the high transcription of ribosomal genes in the nucleoli and its more duplex-like character [12] for its excision of U. TDGFL and TDG82−308 exhibit biphasic kinetics on the NCP, suggesting that SMUG1 and TDG are more similar to each other than to UDG. For both TDGFL and TDG82−308, kobs-fast does not differ significantly from kobs for duplex, suggesting that these rates may correspond to the same kinetic step. Additionally, the rates obtained for TDGFL and for TDG82−308 are not statistically different. Again, we cannot rule out that a small amount of contaminating duplex contributes to the product yield for the fast phase, or, in contrast, this phase corresponds to an NCP population readily accessible to TDG. Recent molecular dynamic simulations examining the steps after binding and up to but excluding bond cleavage have indicated that the rate-limiting step for TDG on duplex substrates is the intercalation of R275 to plug the hole left by the extruded nucleobase (59, 60). Due to the decreased dynamics of DNA in the dyad region (47, 54), there may be a subset of TDG-NCP complexes for which intercalation of R275 is slowed, and thus requires a conformational change in the NCP in order to proceed.