In vivo stability of the Umu mutagenesis proteins: a major role for RecA

Abstract

The Escherichia coli Umu proteins play critical roles in damage-inducible SOS mutagenesis. To avoid any gratuitous mutagenesis, the activity of the Umu proteins is normally kept to a minimum by tight transcriptional and posttranslational regulation. We have, however, previously observed that compared with an isogenic recA ؉ strain, the steady-state levels of the Umu proteins are elevated in a recA730 background (R. Woodgate and D. G. Ennis, Mol. Gen. Genet. 229:10-16, 1991). We have investigated this phenomenon further and find that another coprotease-constitutive (recA*) mutant, a recA432 strain, exhibits a similar phenotype. Analysis revealed that the increased steady-state levels of the Umu proteins in the recA* strains do indeed reflect an in vivo stabilization of the proteins. We have investigated the basis for the phenomenon and find that the mutant RecA* protein stabilizes the Umu proteins by not only converting the labile UmuD protein to the much more stable (and mutagenically active) UmuD protein but by directly stabilizing UmuD itself. In contrast, UmuC does not appear to be directly stabilized by RecA* but is instead dramatically stabilized in the presence of UmuD. On the basis of these observations, we suggest that formation of a UmuDC-RecA*-DNA quaternary complex protects the UmuDC proteins from proteolytic degradation and as a consequence helps to promote the switch from error-free to error-prone mechanisms of DNA repair.

Managing DNA polymerases: Coordinating DNA replication, DNA repair, and DNA recombination

“…When umuDC-dependent TLS was subsequently reconstituted by using purified proteins, the test of biological relevancy was again satisfied when it was found that TLS by Pol V had strict requirement for RecA (6,7,89). It has been suggested that interactions of the UmuDЈ 2 C complex with the end of RecAssDNA filaments position Pol V for its role in the TLS responsible for SOS mutagenesis (90)(91)(92)(93); this positioning seems most likely to involve a direct interaction between UmuDЈ and RecA (90,92,94). It thus appears that this interaction between RecA positioned at a site of DNA damage and UmuDЈ, a component of Pol V, may represent an example of how one polymerase is selectively directed to a particular class of primer termini despite the simultaneous presence of several other DNA polymerases.…”

Genetic Analysis of Repair and Damage Tolerance Mechanisms for DNA-Protein Cross-Links in Escherichia coli

“…The recombination removed the Kan r cassette and produced the ⌬dinB strain (YG6162). To construct YG6168, ⌬(umuDC)596::ermGT was transferred to AB1157 using the DE2302 and EC8 strains as described previously for two-step P1 transduction (19,78). YG6171 (a dinB umuDC double mutant) was constructed by introduction of ⌬dinB::Kan r into YG6168, followed by removal of the Kan r cassette as described above.…”

Escherichia coliDNA Polymerase III Can Replicate Efficiently past a T-Tcis-synCyclobutane Dimer if DNA Polymerase V and the 3′ to 5′ Exonuclease Proofreading Function Encoded bydnaQAre Inactivated

“…1A). To demonstrate that the ⌬dinB61::ble substitution allele is nonpolar, reverse (6), and RW532 do not normally contain FЈ episomes, but for the M13 experiments described herein, they carry the FЈIQ from DH5␣FЈIQ.…”