Purification of a soluble UmuD'C complex from Escherichia coli. Cooperative binding of UmuD'C to single-stranded DNA.
The Escherichia coli UmuD' and UmuC proteins play essential roles in SOS-induced mutagenesis. Previous studies investigating the molecular mechanisms of mutagenesis have been hindered by the lack of availability of a soluble UmuC protein. We report the extensive purification of a soluble UmuD'C complex and its interactions with DNA. The molecular mass of the complex is estimated to be 70 kDa, suggesting that the complex consists of one UmuC (46 kDa) and two UmuD' (12 kDa) molecules. In contrast to its inability to bind to double-stranded DNA, UmuD'C binds cooperatively to single-stranded DNA as measured by agarose gel electrophoresis and confirmed by steady-state fluorescence depolarization. A Hill coefficient, n = 3, characterizes the binding of UmuD'C to M13 DNA and to a 600 nucleotide DNA oligomer, suggesting that at least three protein complexes may interact cooperatively when binding to DNA. The apparent equilibrium binding constant of UmuD'C to single-stranded DNA is approximately 300 nM. Binding of the complex to a short, 80 nucleotide, DNA oligonucleotide was detectable by fluorescence depolarization, but it did not appear to be cooperative. Binding of UmuD'C to single-stranded M13 DNA causes an acceleration of the protein-DNA complex, suggesting that the longer DNA may undergo compaction. The UmuD'C complex associates with RecA-coated DNA, and the UmuD'C complex remains bound to DNA in the presence of RecA.
Bruck
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I
Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles 90089-1340, USA.
Woodgate
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R
McEntee
K
K
Goodman
M F
MF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D017104:Bacteriophage M13 / Q000235:genetics; D002850:Chromatography, Gel; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C078202:UmuC protein, E coli; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli