Abstract
The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
The Escherichia coli Rep helicase is a stable mon- ity of either ss- or ds-DNA to the free protomer of a omer (Mr= 72,802) in the absenceof DNA; half-saturated Rep dimer is clearly influenced by the binding of single-stranded (8s) or duplex(ds) DNA conformation ofDNA bound to the first protomer
Effects of DNA Sequence and Base Composition on Rep-ssDNA Interactions-The E. coli Rep helicase is a nonspecific DNA-binding protein that can unwind duplex DNA independent of its sequence.We examined the interactions of Rep with several 16-nucleotide long ss-oligodeoxynucleotidesin order to determine the effect of base composition on the Rep equilibrium binding and dimerization constants.Three of these, d(pT),s, d(p&, and ~ ( P C )ar~e~ho, mo-oligodeoxynucleotides and a fourth, KL3, has amixed sequence with 50% GC content
Plexes, with the ultimate goal of determining whether these might be influenced by ATP binding and subsequent hydrolysis. For this purpose we have developed a statistical thermodynamic model that describes DNA binding to Rep monomers and thecoupled DNA-induced Rep dimerization
Summary
Reagents and Buffers-All solutions were made with reagent grade chemicals using Milli-Q H20, i.e. distilled H20 that was subsequently deionized and further purified through a Milli-Q Water Purification System (Millipore Corporation, Bedford, MA). Since the radioactivity retained by the DEAE filter is nated by installing an inverted top plate below the membrane, such directly related to the amount of free DNA, it provides a more that each sample well has an O-ring seal above and below the accurate parameter for the determination of nonspecific background membrane By using this modified apparatus, anentire binding isotherm background DNA retained on the nitrocellulose has been determined consisting of a duplicate ortriplicate set of concentration by performing a parallel set of titrations in the absence of protein. As soon as thesamples have been drawn of DNA bound nonspecifically to the nitrocellulose and each through the membrane, the wellswere washed immediately with point in the titration would be overcorrected for this background Another 100 pl of ice-cold binding buffer.
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