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Intermolecular cleavage by UmuD-like mutagenesis proteins
J P McDonald1, E G Frank, A S Levine
1Section on DNA Replication, Repair, and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2725, USA.
Summary
The UmuD-like protein self-cleavage reaction is intermolecular, not intramolecular. This RecA-dependent process requires two separate plasmids and restores mutagenesis in E. coli.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- Microbiology
Background:
- Protein activity is often regulated by self-processing reactions.
- LexA and lambdaCI repressors undergo intramolecular cleavage.
- UmuD-like proteins are involved in mutagenesis.
Purpose of the Study:
- To investigate the mechanism of UmuD-like protein self-processing.
- To determine if UmuD-like protein cleavage is intramolecular or intermolecular.
- To elucidate the role of RecA protein in this process.
Main Methods:
- Mutagenesis of UmuD-like proteins at cleavage and catalytic sites.
- Introduction of mutated plasmids into E. coli.
- In vitro cleavage assays using biotinylated UmuD active site mutant.
Main Results:
- Cleavage of UmuD-like proteins requires two separate plasmids, indicating an intermolecular reaction.
- The intermolecular cleavage is dependent on the RecA protein.
- This process restores cellular mutagenesis in nonmutable E. coli strains.
- Wild-type UmuD' protein can act as an enzyme in vitro.
Conclusions:
- UmuD-like protein self-processing occurs via an intermolecular mechanism.
- RecA protein is essential for this intermolecular cleavage.
- The UmuD' protein can function as a classical enzyme, catalyzing intermolecular cleavage.