Intermolecular cleavage by UmuD-like enzymes: identification of residues required for cleavage and substrate specificity 1 1Edited by A. Gottesman

The Preferred Substrate for RecA-Mediated Cleavage of Bacteriophage 434 Repressor Is the DNA-Bound Dimer

“…Several lines of evidence indicate that the monomeric forms of LexA and repressor proteins are the preferred targets for the RecAstimulated autocleavage reaction (15,18,27). In contrast, it is the dimeric form of UmuD that undergoes efficient autocleavage via an intermolecular pathway (23,24). These findings show that both mono-and dimeric forms of the self-cleaving proteins can undergo RecA-stimulated autocleavage Previous studies showed that the presence of substoichiometric amounts of nonspecific or specific DNA stimulates the complete conversion of 434 repressor into oligomers (6,7).…”

The Bacteriophage 434 Repressor Dimer Preferentially Undergoes Autoproteolysis by an Intramolecular Mechanism

“…To determine whether the 434 repressor utilizes an inter-or intramolecular autocleavage mechanism, we used an approach similar to the approach used by Kim and Little (20) and McDonald et al (28,29) and created two repressor mutants having substitutions in the residues that comprise part of either the active (or "catalytic") site of the 434 repressor (S126) or its cleavage site (G89). We examined the ability of each protein to undergo autocleavage alone, in combination with the other protein, or in the presence of the wild-type repressor.…”

“…UmuD and MucA proteins having mutations at residues homologous to S126 and G89 are incapable of undergoing RecA*-stimulated autocleavage (29). However, cleavage of UmuD and MucA can take place when a subunit having a mutation in the active site is mixed with a subunit having a mutation in the cleavage site (29).…”

“…RecA*-mediated autoproteolysis in vivo was monitored using a method similar to the method described by McDonald et al (28,29). E. coli X-90 (7), transformed with the desired repressor-expressing plasmid(s), was grown to the mid-log phase (ϳ1.0 ϫ 10 8 CFU/ml) in LB medium supplemented with the appropriate antibiotic(s).…”

“…Therefore, normally, the active site residues of one subunit of LexA and the repressor catalyze cleavage of the linker located within the same subunit; that is, in these proteins, cleavage must occur intramolecularly (16,17,22,34,36). UmuD undergoes cleavage as a dimer, and in the presence of RecA, it uses an intermolecular cleavage strategy (28,29). In this case, one monomer of the UmuD dimer cleaves in the linker of its partner subunit (43).…”