Evidence for multiple active forms of the DNA damage response protein UmuD

Abstract

All organisms experience DNA damage and have evolved multiple pathways to maintain genomic stability. In Escherichia coli, the SOS response to DNA damage involves the induction of 57 genes including the umuD gene products. The UmuD homodimer of 139‐amino acid subunits binds a RecA/ssDNA nucleoprotein filament which stimulates the latent ability of UmuD to cleave its N‐terminal 24‐amino acids, producing UmuD′2. UmuD2 and UmuD′2 play differential roles in the DNA damage response. Several models have been proposed for the conformations of UmuD2 including two versions with the N‐terminal arms in trans and two versions with the arms in cis (Beuning, et al. 2006 J. Biol. Chem 281:9633). These conformations expose different binding surfaces that may interact with other proteins. The goal of our research is to determine the conformation and dynamics of the umuD gene products in order to understand how their differential protein‐protein interactions regulate cellular responses to DNA damage. Our preliminary results suggest that a single amino acid substitution, N41G, weakens the KD for dimerization by over five orders of magnitude. This N41G mutation yields a UmuD monomer that is cleavable in the presence and absence of RecA/ssDNA. These results suggest that UmuD2 exists in multiple active conformations and that the cis conformation may be physiologically relevant. Supported by NSF Career Award MCB‐0845033.