DNA damage-inducible mutagenesis in Escherichia coli is largely dependent upon the activity of the

Most SOS mutagenesis in Escherichia coli is dependent on the UmuD and UmuC proteins. Perhaps as a consequence, the activity of these proteins is exquisitely regulated. The intracellular level of UmuD and UmuC is normally quite low but increases dramatically in lon − strains, suggesting that both proteins are substrates of the Lon protease. We report here that the highly purified UmuD protein is specifically degraded in vitro by Lon in an ATP-dependent manner. To identify the regions of UmuD necessary for Lon-mediated proteolysis, we performed 'alanine-stretch' mutagenesis on umuD and followed the stability of the mutant protein in vivo. Such an approach allowed us to localize the site(s) within UmuD responsible for Lon-mediated proteolysis. The primary signal is located between residues 15 and 18 (FPLF), with an auxiliary site between residues 26 and 29 (FPSP), of the amino terminus of UmuD. Transfer of the amino terminus of UmuD (residues 1-40) to an otherwise stable protein imparts Lon-mediated proteolysis, thereby indicating that the amino terminus of UmuD is sufficient for Lon recognition and the ensuing degradation of the protein.

The Escherichia coli UmuD′ protein is a subunit of the recently described error‐prone DNA polymerase, pol V. UmuD′ is initially synthesized as an unstable and mutagenically inactive pro‐protein, UmuD. Upon processing, UmuD′ assumes a relatively stable conformation and becomes mutagenically active. While UmuD and UmuD′ by themselves exist in vivo as homodimers, when together they preferentially interact to form heterodimers. Quite strikingly, it is in this context that UmuD′ becomes susceptible to ClpXP‐mediated proteolysis. Here we report a novel targeting mechanism designed for degrading the mutagenically active UmuD′ subunit of the UmuD/D′ heterodimer complex, while leaving the UmuD protein intact. Surprisingly, a signal that is essential and sufficient for targeting UmuD′ for degradation was found to reside on UmuD not UmuD′. UmuD was also shown to be capable of channeling an excess of UmuD′ to ClpXP for degradation, thereby providing a mechanism whereby cells can limit error‐prone DNA replication.