Escherichia coli DNA repair helicase Lhr is also a uracil‐DNA glycosylase

DNA glycosylases protect genetic fidelity during DNA replication by removing potentially mutagenic chemically damaged DNA bases. Bacterial Lhr proteins are well‐characterized DNA repair helicases that are fused to additional 600–700 amino acids of unknown function, but with structural homology to SecB chaperones and AlkZ DNA glycosylases. Here, we identify that Escherichia coli Lhr is a uracil‐DNA glycosylase (UDG) that depends on an active site aspartic acid residue. We show that the Lhr DNA helicase activity is functionally independent of the UDG activity, but that the helicase domains are required for fully active UDG activity. Consistent with UDG activity, deletion of lhr from the E. coli chromosome sensitized cells to oxidative stress that triggers cytosine deamination to uracil. The ability of Lhr to translocate single‐stranded DNA and remove uracil bases suggests a surveillance role to seek and remove potentially mutagenic base changes during replication stress. Bacterial Large helicase‐related (Lhr) DNA repair proteins have well‐characterized helicase domains that translocate DNA, but in addition possess large (800 amino acid) C‐terminal domains of unknown function, which we show have uracil‐DNA glycosylase activity. We identify a novel active site for this function, and that loss of Lhr in Escherichia coli sensitizes cells to oxidative stress.