DNA glycosylase activity of E. coli Lhr is independent from its DNA helicase activity
Full length Lhr was substantially more active than Lhr-CTD as a uracil-DNA glycosylase when measured in assays as a function of time (Figure 3A)  this may be explained by much more stable DNA binding by full length Lhr compared with Lhr-CTD that was observed in EMSAs (Figure 2A). We therefore continued to use full-length Lhr to further investigate uracil-DNA glycosylase function against flayed duplex DNA molecules that are substrates for unwinding by the Lhr 3’ to 5’ DNA helicase activity [11]. For this work the duplex substrate was formed from annealing uracil-containing ssDNA with its unmodified complementary DNA strand, with uracil positioned 8nt from the fork branchpoint, 18 nt from the Cy5-DNA 5’ end. Measured as a function of time, Lhr generated glycosylase product from the uracil duplex at least 5-fold more effectively than when incubated with uracil-ssDNA (Figure 3B ), and Lhr was more active than Lhr-CTD on the uracil-fork DNA (Figure 3C ). Neither Lhr or Lhr-CTD gave any glycosylase product when uracil was substituted for a single 8-oxoguanine residue at the same position in DNA (Figure 3D ). The product of Lhr from uracil-DNA single strands or duplex migrated close to the 16 nt marker, indicating that the same glycosylase product was formed from both substrates. Glycosylase assay conditions in Figures 1-3 included Mg2+ in the reaction buffer, but Lhr was also active in buffers containing EDTA, Mn2+ and Ca2+ instead of Mg2+ (Figure 3E lanes 1-4) but was inactive as a glycosylase on DNA lacking a uracil residue (Figure 3E lanes 5-8). LhrD1536 that is inactive as a uracil-DNA glycosylase was proficient at fork DNA unwinding (Figure 3F ), and we therefore conclude that the uracil glycosylase activity of Lhr is functionally distinct from helicase activity, but we observe that glycosylase activity is enhanced when the helicase domains are present, by contributing to DNA binding (Figure 2A ).