Quantitative Characterization of Interactions of the Escherichia Coli SOS DNA Damage Response Proteins UmuD and UmuD' with the Replicative DNA Polymerase

Abstract

The Y family of DNA polymerases is a group of DNA damage tolerance enzymes with the specialized ability to bypass DNA lesions by inserting nucleotides opposite damaged sites in DNA. Translesion synthesis (TLS) responsible for most of the mutagenesis induced by UV radiation requires the UmuD'2 protein (the cleaved form of the UmuD2), UmuC, and RecA. UmuD'2 activates UmuC, the catalytic subunit of the Y family DNA polymerase V, for mutagenic DNA replication. UmuD2 and UmuD'2 make a remarkable number of specific protein-protein contacts to DNA polymerases. Despite the nearly identical primary structure of UmuD2 and UmuD'2, their interactions with the same partner can differ in affinity and functional significance. Analysis of the UmuD/UmuD'-pol III interactions by affinity chromatography indicated that UmuD has a lower affinity for alpha (α) subunit of E. coli’s replicative polymerase III than does UmuD'. We aim to understand how binding of UmuD dimers is coordinated with the activity of α subunit. We are utilizing the biochemical and biophysical methods to look at the kinetics of α subunit activity and at the formation of α-UmuD2 and α-UmuD'2 complexes. We are characterizing the ability of the α subunit to copy both damaged and undamaged DNA in the presence of UmuD and UmuD'. In order to quantify the binding of UmuD and UmuD' to the α subunit, we are determining the Kd (equilibrium dissociation constant) for this interaction by measuring intrinsic tryptophan fluorescence of the α subunit.