Targeting of the UmuD, UmuD', and MucA' mutagenesis proteins to DNA by RecA protein.
In addition to its critical role in genetic recombination, the Escherichia coli RecA protein plays a pivotal role in SOS-induced mutagenesis. This role can be separated genetically into three steps: (i) depression of the SOS regulon by mediating the posttranslational cleavage of the LexA repressor, (ii) activation of UmuD'-like proteins by mediating cleavage of the UmuD-like proteins, and (iii) a direct step, possibly to interact with and to target the Umu-like mutagenesis proteins to lesions in DNA. We have analyzed RecA's third role biochemically using protein affinity chromatography and an agarose-based DNA mobility-shift assay. RecA730 protein from a crude cell extract was specifically retained on UmuD and UmuD' protein affinity columns, suggesting that these proteins physically interact. Normally, neither UmuD nor UmuD' shows any affinity for DNA. In the presence of RecA protein, however, UmuD and UmuD' were targeted to DNA. RecA1730 protein, which is defective for UmuD' but proficient for MucA'-promoted mutagenesis, showed a dramatically reduced capacity to target UmuD' to DNA but was able to target a significant portion of MucA' to DNA. These data support the suggestion that the direct role of RecA protein in SOS-induced mutagenesis is to interact with and target the Umu-like mutagenesis proteins to DNA.
Frank
E G
EG
Section on DNA Replication, Repair, and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Hauser
J
J
Levine
A S
AS
Woodgate
R
R
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D004269:DNA, Bacterial / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D011938:Rec A Recombinases / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C063757:MucA protein, Bacteria; D011994:Recombinant Proteins; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli