Translesion synthesis protein ImuA from Mycolicibacterium smegmatis is a hexameric helicase-nuclease

Translesion DNA synthesis (TLS) enables DNA replication across damaged DNA and promotes stress-induced mutagenesis that contributes to antibiotic resistance in bacteria. The conserved ImuABC mutasome is essential for TLS in many bacterial species, yet the molecular function of its accessory protein, ImuA, has remained elusive. Here we show that Mycolicibacterium smegmatis ImuA assembles into a hexameric complex, likely arranged as a dimer of trimers, with dual enzymatic activities that reshape current models of its role in DNA damage tolerance. We show that ImuA functions as an ATP-dependent helicase that preferentially unwinds DNA substrates containing single-stranded DNA overhangs and identify amino acids required for both hexamer formation and helicase activity. Unexpectedly, ImuA also possesses ATP-independent 5' exonuclease activity, selectively processing ssDNA substrates with free 5' ends. We show a basic patch on the N-terminus is essential for stabilizing both the nuclease motif and oligomerization. Together, these findings identify ImuA as an active DNA-processing enzyme rather than a passive accessory factor and establish oligomerization as a prerequisite for its function. Our work provides a mechanistic framework for understanding how ImuA may function within the ImuABC mutasome to coordinate DNA processing during translesion synthesis.