UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD'.

Abstract

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli produces a marked elevation in mutation rate. The mutation pathway is a component of the induced, multigene SOS response. SOS mutagenesis is a tightly regulated process dependent on two RecA-mediated proteolytic events: cleavage of the LexA repressor to induce the UmuC and UmuD mutagenesis proteins, and cleavage of UmuD to UmuD9 to activate the mutation pathway. To investigate the protein-protein interactions responsible for SOS mutagenesis, we have studied the interaction of UmuC, UmuD, and UmuD9. To probe intracellular interaction, we have used immunoprecipitation techniques with antibodies against UmuC or UmuD and UmuD9. We have found that antibody to UmuC precipitates UmuD9 from cell extracts, and antibody to UmuD and UmuD9 precipitates UmuC. Thus we conclude that UmuC probably associates tightly with UmuD9 in cells. For biochemical studies, we have purified the UmuC and UmuD9 proteins to use with the previously purified UmuD. UmuC associates strongly with an affinity column of UmuD and UmuD9, eluting only under strongly dissociating conditions (2 M urea or 1.5 M KSCN). UmuC also associates efficiently with UmuD or UmuD9 in solution, as judged by velocity sedimentation in a glycerol gradient. The likely stoichiometry is one UmuC with a dimeric UmuD or UmuD9. From these experiments and previous work, we infer that SOS mutagenesis depends on the action of the UmuC-UmuD9 complex and probably RecA to rescue a stalled DNA polymerase III holoenzyme at the DNA lesion.