2013 • Physical and LIfe Sciences
Characterization of the N-terminal arms of the polymerase manager protein UmuD
Lead Presenter: David Murison
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.