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 S OS-induced mutagenesis, Previous studies investigating the molecular m echanisms of mutagenesis have been hindered by the lack of availabilit y of a soluble UmuC protein, We report the extensive purification of a soluble UmuD'C complex and its interactions with DNA, The molecular m ass of the complex is estimated to be 70 kDa, suggesting that the comp lex 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 bind s cooperatively to single-stranded DNA as measured by agarose gel elec trophoresis 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 th ree 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 nu cleotide, DNA oligonucleotide was detectable by fluorescence depolariz ation, but it did not appear to be cooperative, Binding of UmuD'C to s ingle stranded M13 DNA causes an acceleration of the protein-DNA compl ex, suggesting that the longer DNA may undergo compaction, The UmuD'C complex associates with RecA-coated DNA, and the UmuD'C complex remain s bound to DNA in the presence of RecA.