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. æFull-length UmuD is expressed as a 139-amino-acid protein, which eventually cleaves its N-terminal 24 amino acids to form UmuDÍ. ææBoth UmuD and UmuDÍ exist alone as homodimers, but can also exchange to form UmuDDÍ heterodimers. æThe N-termini of UmuD exist as conformationally-dynamic arms, and contain a number of substrate recognition sites. æTherefore, cleavage of UmuD to UmuDÍ dramatically affects the function of the protein. Recently, we have constructed additional N-terminally truncated versions of UmuD. æUmuD 8 (UmuD ?1-7) and UmuD 18 (UmuD ?1-17) were used as tools to study the conformation of the N-terminal arms, the effect on cleavage as well as the 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. Although UmuD 8 is cleaved as efficiently as full-length UmuD in vitro and in vivo, UmuD 18 does not cleave to form UmuD?. We have also determined that UmuD 8 is proficient for UV mutagenesis, but intriguingly, does not confer resistance to UV irradiation in a ?recJ strain. æWe are utilizing site-directed spin labeling and electron paramagnetic resonance to characterize the local motion of the N-terminal arms of the different UmuD variants.