Characterization of the N‐terminal Arms of the Polymerase Manager Protein UmuD

Abstract

Escherichia coli cells that are exposed to exogenous or endogenous DNA damaging agents invoke the SOS response that involves expression of the umuD gene products. Full-length UmuD is expressed as a 139-amino-acid protein, which eventually cleaves its N-terminal 24 amino acids to form UmuD′. Both UmuD and UmuD′ exist alone as homodimers, but can also exchange to form UmuDD′ heterodimers. The N-termini of UmuD exist as conformationally-dynamic arms, and contain a number of recognition sites for partner proteins. Cleavage of UmuD to UmuD′ dramatically affects the function of the protein, and activates UmuC for translesion synthesis (TLS). We have constructed additional N-terminal truncations of UmuD, UmuD 8 (UmuD Δ1-7) and UmuD 18 (UmuD Δ1-17), and are probing the conformation of the N-terminal arms, their effect on cleavage as well as their effect on protein-protein interactions. We found that the loss of just the N-terminal seven amino acids of UmuD results in significant changes in conformation of the N-terminal arms and in relative preference for binding sites on the partner protein DNA polymerase III α subunit. UmuD 8 is cleaved as efficiently as full-length UmuD in vitro and in vivo, but expression of plasmid-borne UmuD 8 makes cells hypersensitive to UV irradiation. UmuD 18 does not cleave to form UmuDʹ, but confers resistance to UV irradiation. We used site-directed spin labeling for analysis by electron paramagnetic resonance (EPR) and covalent cross-linking to characterize the local motion of the N-terminal arms of the different UmuD variants. This investigation will lead to understanding the mechanism by which UmuD 8 causes UV hypersensitivity. This work is supported by the National Science Foundation and American Cancer Society.