Discussion
We provide biochemical evidence that E. coli Lhr is a uracil-DNA
glycosylase (Lhr-UDG), a new function for bacterial Lhr proteins
alongside their well-characterized 3’ to 5’ single DNA translocation
activity that is stimulated by forked or flayed DNA substrates [5, 7,
11]. We show that the Lhr-UDG activity requires an active site
aspartate residue (Asp-1536), similarly to the active site aspartate
general base (Asp-62) that is essential for major groups of UNG/UDG
proteins [22]. The Lhr UDG function is positioned in the previously
uncharacterized Lhr-CTD though this fragment of Lhr was proficient as
a ‘stand-alone’ uracil-DNA glycosylase, its activity was significantly
increased by the presence of the Lhr helicase domains, probably by the
helicase domains providing more stable DNA binding compared with
Lhr-CTD, observed in EMSAs. Inactivating the Lhr-UDG activity did not
inactivate DNA unwinding by Lhr, providing further support for the DNA
binding functions of Lhr being concentrated in the helicase domains.
Loss of Lhr from bacterial cells (Δlhr ) causes mild sensitivity
to AZT [14], a phenotype we also observed after generating
Δlhr cells and removing the inserted antibiotic resistance
marker. We identified that Δlhr cells were also significantly
more sensitive than lhr+ cells to oxidative
stress induced by hydrogen peroxide, which is one of several routes
causing genetic damage by cytosine deamination in bacterial cells. This
therefore supports our in vitro observations of Lhr-UDG function.
UDGs are ubiquitous in nature, although this is the first report of a
UDG fused to a DNA helicase. E. coli has a canonical UDG enzyme
that functions in global DNA repair coupled with stable DNA replication
upregulation of Mycobacterial Lhr in response to mitomycin C treatment
[15], and the sensitivity of E. coli cells to the polymerase
inhibitor AZT when they lack lhr , may indicate that Lhr is
activated as part of bacterial responses to specific forms of
replication-stress. In this context removal of uracil from DNA by Lhr
may protect genetic fidelity at sites that are overcoming blocked DNA
replication. We cannot exclude that Lhr may be able to remove other
lesions or chemical modifications from DNA, although we observed that it
was inactive as a glycosylase against 8-oxoguanine, suggesting that is
has at least specificity for recognizing pyrimidine damage over purines.