Organisms experience DNA damage from numerous sources. When bacterial cells experience DNA damage and other stresses, the SOS response is induced, leading to upregulation of at least 50 genes in E. coli. Among the upregulated genes are specialized Y family DNA polymerases, which replicate damaged DNA and play a role in tolerance to DNA damage. The activity of these potentially mutagenic proteins is tightly regulated. The manager proteins UmuD2 and the cleaved form UmuD'2 provide one regulatory mechanism. The umuD gene products interact with both Y family polymerases, the replicative DNA polymerase, and the beta processivity clamp. The goal of this study is to determine how the conformation and dynamics of the umuD gene products regulate the cellular response to DNA damage. We are characterizing the effects of UmuD and UmuD' on DNA polymerase III activity and probing their conformations using fluorescence resonance energy transfer (FRET) and hydrogen‐deuterium exchange mass spectrometry (HXMS). In HXMS experiments, backbone amide hydrogens that are solvent‐accessible exchange with deuterium over time, whereas inaccessible hydrogens do not exchange. Our HXMS results reveal that the N‐terminal arm of UmuD, which is not present in the cleaved form UmuD′, exhibits local partial unfolding. Additionally, there are substantial regions of stable conformation in both proteins.This work is funded by a Dreyfus Foundation New Faculty Award.