The Binding of fd Gene 5 Protein to Polydeoxynucleotides: Evidence from CD Measurements for Two Binding Modes

Abstract

Circular dichroism measurements were used to study the binding of fd gene 5 protein to fd DNA, to six polydeoxynucleotides (poly[d(A)], poly[d(T)], poly[d(I)], poly[d(C)], poly[d(A-T)], and the random copolymer poly[d(A,T)]), and to three oligodeoxynucleotides (d(pA)20, d(pA)7, and d(pT)7). Titrations of these DNAs with fd gene 5 protein were generally done in a low ionic strength buffer (5 mM Tris-HCl, pH 7.0 or 7.8) to insure tight binding, needed to obtain stoichiometric endpoints. By monitoring the CD of the nucleic acids above 250 nm, where the protein has no significant intrinsic optical activity, we found that there were two modes of binding, with the number of nucleotides covered by a gene 5 protein monomer (n) being close to either 4 or 3. These stoichiometries depended upon which polymer was titrated as well as upon the protein concentration. Single endpoints at nucleotide/protein molar ratios close to 3 were found during titrations of poly[d(T)] and fd DNA (giving n = 3.1 and 2.8 +/- 0.2, respectively), while CD changes with two apparent endpoints at nucleotide/protein molar ratios close to 4 and approximately 3 were found during titrations of poly[d(A)], poly[d(I)], poly[d(A-T)], and poly[d(A,T)] (with the first endpoints giving n = 4.1 4.0, 4.0, and 4.1 +/- 0.3, respectively). Calculations showed that the CD changes we observed during these latter titrations were consistent with a switch between two non-interacting binding modes of n = 4 and n = 3. We found no evidence for an n = 5 binding mode. One implication of our results is that the Brayer and McPherson model for the helical gene 5 protein-DNA complex, which has 5 nucleotides bound per protein monomer (G. Brayer and A. McPherson, J. Biomol. Struct. and Dyn. 2, 495-510, 1984), cannot be correct for the detailed solution structure of the complex. We interpreted the CD changes above 250 nm upon binding of the gene 5 protein to single-stranded DNAs to be the result of a slight unstacking of the bases, along with a significant alteration of the CD contributions of the individual nucleotides in the case of A-and/or T-containing DNAs. Interestingly, CD contributions attributed to nearest-neighbor interactions in free poly[d(A-T)], poly[d(A,T)], poly[d(A)], and poly[d(T)] were partially maintained in the CD spectra of the protein-saturated polymers, so that neighboring nucleotides, when bound to the protein at 20 degrees C, appeared to interact with one another in much the same manner as in the free polymers at 50 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Human replication protein A binds single-stranded DNA in two distinct complexes.

“…The filamentous bacteriophage Pfl gene 5 protein has two distinct modes of ssDNA binding which appear to result from the occupancy of either one or two DNA-binding sites on a gene 5 homodimer by ssDNA (11). Other SSB proteins have somewhat similar properties (10,29 (Fig. 8A).…”

Electrostatic potential distribution of the gene V protein from Ff phage facilitates cooperative DNA binding: A model of the GVP‐ssDNA complex

“…More detailed studies regarding the interactions between GVP and DNA have been carried out (reviewed in Kansy et al, 1986). In particular, NMR and fluorescence spectroscopies have implicated several amino acids of GVP that are in contact with DNA in the GVP-ssDNA complex (King & Coleman, 1987Dick et al, 1989;Stassen et al, 1992;Folkers et al, 1993).…”

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

“…Recently, a number of groups have reported observations of specific noncovalent complexes of biomolecules from solution using ESI-MS (6-11). These reports prompted us to investigate the application of this technique to intact protein-DNA noncovalent complexes.We chose gene V protein from bacteriophage fl for our study due to its well characterized structure (12-16) and the extensive studies on the DNA binding stoichiometry and cooperatively (17)(18)(19)(20)(21)(22). The gene V protein stabilizes singlestranded DNA (ssDNA) during phage replication, and shifts the synthesis of phage DNA from double-stranded form to single-stranded form, in the.…”

“…We chose gene V protein from bacteriophage fl for our study due to its well characterized structure (12)(13)(14)(15)(16) and the extensive studies on the DNA binding stoichiometry and cooperatively (17)(18)(19)(20)(21)(22). The gene V protein stabilizes singlestranded DNA (ssDNA) during phage replication, and shifts the synthesis of phage DNA from double-stranded form to single-stranded form, in the.…”