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.