Regulation of UmuD cleavage: role of the amino-terminal tail.
An essential step in SOS mutagenesis is the RecA-mediated posttranslational processing of UmuD-like proteins to the shorter, but mutagenically active, UmuD'-like proteins. Interestingly, the UmuD-like proteins undergo posttranslational processing at different rates. For example, although the Escherichia coli UmuD (UmuDEc) and the Salmonella typhimurium UmuD (UmuDSt) proteins are 73% identical, UmuDSt is processed in vivo at a significantly faster rate than the UmuDEc protein. Here, we report experiments aimed at investigating the molecular basis of these phenotypic differences. The faster rate of UmuDSt cleavage probably does not result solely from a better interaction with RecA, since we observed that, in vitro, UmuDSt undergoes RecA-independent autocatalytic processing about four-times faster than UmuDEc. By constructing chimeric UmuD proteins, we determined that the amino-terminal tail of the UmuD proteins proximal to the Cys24-Gly25 cleavage site is mainly responsible for the difference in UmuDSt and UmuDEc cleavage rates. Site-directed mutagenesis of the UmuDEc protein suggests that most of the enhanced cleavage observed with the UmuDSt protein can be attributed to the presence of a Pro23 residue, juxtaposed to the cleavage site in UmuDSt. Furthermore, this proline residue appears to result in a UmuD protein that is a much better substrate for intermolecular cleavage. These findings clearly implicate the N-terminal tail of the UmuD-like proteins as playing an important and unexpected regulatory function in the maturation of the mutagenically active UmuD'-like mutagenesis proteins.
McDonald
J P
JP
Section on DNA Replication Repair, and Mutagenesis, National Institute of Child Health and Human Development, Bethesda, MD, 20892-2725, USA.
Maury
E E
EE
Levine
A S
AS
Woodgate
R
R
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011392:Proline / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D011392:Proline; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli