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 ).