Fab fragments from hybridoma HEd 10 [Lee, J. S., Lewis, J.R., Morgan, A.R., Mosmann, T.R., & Singh, B. (1981) Nucleic Acids Res. 9, 1707-1721] were prepared in large amounts by papain digestion of the immunoglobulin G (IgG) fraction from ascites fluid. Binding data were generated by a fluorescence quenching technique, and binding constants [K(0)] were estimated from Scatchard plots. The Fab fragments bound tightly to poly(dT) [K(0) = 12.7 X 10(6) M-1], and analysis of binding constants for the series p(dT)2 to p(dT)17 showed that the recognition sequence consisted of four consecutive residues. The effect of ionic strength on the interaction suggested that only two phosphates were involved. Binding constants for poly(dU), poly[d(brU)], poly[d(brC)], and poly(rU) were 1.0 X 10(6) M-1, 18.8 X 10(6) M-1, 0.5 X 10(6) M-1, and less than 0.5 X 10(6) M-1, respectively, implicating the involvement of the 3, 4, and 5 positions of the pyrimidine ring as well as the deoxyribose sugar in the recognition process. At high ionic strength (0.5 M) K(0) for whole IgG binding to poly(dT) was 75 X 10(6) M-1 compared to a value of 1.1 X 10(6) M-1 for the Fab fragment. These results may have implications for the tissue damage caused by DNA-containing immune complexes in systemic lupus erythematosus.
Ethidium fluorescence assays were adapted for the rapid and sensitive detection of precA; in addition, fluorescence measurements on binding precA to linear, OC and CCC PM2 DNAs have enabled the stoichiometry of precA binding as well as the precA-induced unwinding angle of DNA to be determined. The stoichiometry of binding was independently confirmed by sedimentation analysis to be one precA molecule per 3 bp. The unwinding angle was also independently confirmed by measurements of fluorescence changes induced by the binding of precA to CCC DNA which was relaxed by topoisomerase to give a precA-induced unwinding angle of 51 degrees. Electron microscopy of OC DNA molecules which bound nonsaturating amounts of precA revealed that the length increase in DNA due to precA was approximately 55%. Finally, examination of negatively stained precA complexes with a variety of linear DNAs showed that the minor groove is the primary site of interaction for this protein.
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