br Materials and methods br Results br Discussion All

Materials and methods
Results
Discussion All the UDG superfamily glycosylases examined here, UDG, SMUG1, TDGFL, and TDG82−308, are capable of completely converting U-containing duplex substrates to product, though at different rates. Under STO conditions, kobs reflects the slowest kinetic step up to and chemistry. These steps include DNA binding, distortion of the DNA helix for base flipping into the active site, intercalation of an amino gstp1 residue into the DNA helix to plug the resulting hole, and chemistry. In comparing the three UDG superfamily glycosylases excising U from U:G in duplex, it is clear that UDG is the fastest, while SMUG1, TDGFL, and TDG82−308 all have comparable kobs that are ∼3-12 times slower than UDG. That UDG is fastest then begs the question as to whether SMUG1 and TDG serve functions only redundant to UDG for excision of U in duplex-like DNA, that is, DNA that is not wrapped around histone proteins. Duplex-like DNA may be found in the cell in several situations: between adjacent nucleosomes, during chromatin remodeling, or transiently unwrapped from nucleosomes during transcription and replication. The cell cycle regulation of each of the UDG superfamily glycosylases may suggest which of these types of duplex-like DNA, if any, are substrates for the enzymes. UDG is upregulated during S-phase and tends to accumulate specifically at replication foci to remove U misincorporated from the dNTP pool (6). UDG may exploit the existence of unwrapped DNA at this time to maximize excision of U from duplex-like or even ssDNA. SMUG1, on the other hand, does not undergo appreciable variation of expression throughout the cell cycle, and instead is expressed continuously at low levels, in both actively replicating and quiescent cells (6). It is possible that SMUG1, with observed higher activity on duplex substrates, may take advantage of transient duplex-like DNA throughout the cell cycle. Like UDG, TDG levels are regulated, but TDG is actively degraded at the G1/S transition [34,35]. Therefore, unlike UDG and SMUG1, TDG is not available during replication. Should TDG require duplex-like DNA for efficiency, it would need to rely on other processes, such as transcription or chromatin remodelers, to access its substrate. Though it is clear that UDG superfamily glycosylases are capable of excising U in duplex DNA, their activities on packaged DNA have not been as well characterized. Here we have shown that for an outward-facing U at the dyad axis of an NCP compared to the analogous duplex, UDG is minimally inhibited, SMUG1 is the most inhibited, and TDG exhibits intermediate levels of inhibition. UDG is unique among these three UDG superfamily glycosylases because of its monophasic kinetics and full conversion to product for the NCP substrate. These results are especially interesting given that the UDG enzyme used in this study is the E. coli ortholog, which was not influenced by the presence of nucleosomes through its evolution. We note, however, that the E. coli and human orthologues of UDG are highly similar in both sequence [42] and overall conformation. Comparison of the crystal structures of E. coli UDG and human UNG show root mean square deviation of <1 Å when Cα are aligned [43]. The single-phase kinetics reveal that the entire population of NCP is in a form that is readily accessible to UDG for glycosidic bond cleavage, with a kobs that is only ∼4-times slower than duplex. This observation is consistent with what we and others have observed previously, a slower kobs for UDG excision at the dyad region versus duplex [38,53]. We note, however, that Cole et al. observed a more dramatic decrease in kobs at the dyad than what we describe here [44]. We attribute this discrepancy to a difference in the DNA positioning sequence, namely, the 5S rDNA versus Widom 601 positioning sequences. For the NCP substrates, kobs could represent the same kinetic step as kobs on duplex. However, it is also possible that kobs on the NCP represents a rate-limiting step prior to chemistry.