"Activated"-RecA protein affinity chromatography of LexA repressor and other SOS-regulated proteins.
We have developed an affinity column to study the interaction of LexA repressor and other substrates with the activated form of RecA protein. Nucleoprotein complexes of RecA protein, (dT)25-30, and adenosine 5'-[gamma-S]thio-triphosphate were formed in solution and bound to RecA protein-agarose columns. These "activated"-RecA nucleoprotein complexes were retained by strong hydrophobic interactions. Purified LexA protein bound tightly to these activated RecA columns, whereas the LexA protein bound poorly to RecA-agarose alone. Once bound, LexA protein underwent specific proteolysis, and the fragments were released from the complex. The mutant LexA protein, LexA-SA119, which cannot carry out self-cleavage or RecA-mediated cleavage in solution, bound efficiently to the activated RecA column but was not cleaved, indicating that these columns can be used to identify residues involved in RecA-LexA binding. As an example of this use, nucleoprotein complexes were prepared using the RecA430 protein. In vivo the recA430 mutation blocks induction of the SOS response. LexA protein was not efficiently retained on the immobilized RecA430 complexes, suggesting that Gly-204 is required for efficient repressor binding. These results show that activated RecA affinity columns can be used to investigate the binding and cleaving properties of mutationally altered RecA and LexA proteins. Additionally, these activated RecA columns have been used to investigate binding interactions of phage lambda repressor, as well as the UmuC protein, which is required for chemical mutagenesis.
Freitag
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Laboratory of Biomedical and Environmental Sciences, School of Medicine, University of California, Los Angeles 90024.
McEntee
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K
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics; D002846:Chromatography, Affinity; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D009698:Nucleoproteins / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism*; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013014:SOS Response, Genetics; D012697:Serine Endopeptidases; D014157:Transcription Factors / Q000378:metabolism*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004259:DNA-Directed DNA Polymerase; D029968:Escherichia coli Proteins; D009698:Nucleoproteins; D011938:Rec A Recombinases; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014157:Transcription Factors; D000099160:LexA Repressor Protein; C055218:UmuD protein, E coli