Study of the binding of single-stranded DNA-binding protein to DNA and poly(rA) using electric field induced birefringence and circular dichroism spectroscopy

Abstract

Binding of the single-stranded DNA-binding protein (SSB) of Escherichia coli to single-stranded (ss) polynucleotides produces characteristic changes in the absorbance (OD) and circular dichroism (CD) spectra of the polynucleotides. By use of these techniques, complexes of SSB protein and poly(rA) were shown to display two of the binding modes reported by Lohman and Overman [Lohman, T.M., & Overman, L. (1985) J. Biol. Chem. 260, 3594-3603]. The circular dichroism spectra of the "low salt" (10 mM NaCl) and "high salt" (greater than 50 mM NaCl) binding mode are similar in shape, but not in intensity. SSB binding to poly(rA) yields a complexed CD spectrum that shares several characteristics with the spectra obtained for the binding of AdDBP, GP32, and gene V protein to poly(rA). We therefore propose that the local structure of the SSB-poly(rA) complex is comparable to the structures proposed for the complexes of these three-stranded DNA-binding proteins with DNA (and RNA) and independent of the SSB-binding mode. Electric field induced birefringence experiments were used to show that the projected base-base distance of the complex is about 0.23 nm, in agreement with electron microscopy results. Nevertheless, the local distance between the successive bases in the complex will be quite large, due to the coiling of the DNA around the SSB tetramer, thus partly explaining the observed CD changes induced upon complexation with single-stranded DNA and RNA.

Apparent Heat Capacity Change Accompanying a Nonspecific Protein-DNA Interaction. Escherichia coli SSB Tetramer Binding to Oligodeoxyadenylates

“…Using Raman spectroscopy, Otto et al (1988) observed increased hyperchromicities upon binding of either Ml3 gene V or T4 gene 32 proteins to poly (dA) but not to poly (A). As mentioned above, Kuil et al (1990) have shown using electric field induced birefringence that the bases in poly(dA) become partially unstacked upon binding E. coli SSB protein, and similar observations have been made for T4 gene 32 protein (Scheerhagen et al, 1985;van Amerongen et al, 1988). If coupling of base unstacking to protein-DNA binding provides the explanation for the nonlinear van't Hoff plots observed for the E. coli SSB tetramer-dA(pA)" binding, then nonlinear van't Hoff plots with ACp < 0 should also be observed for binding of poly(dA) to the T4 gene 32 protein, the fl gene V protein, and possibly other SSB proteins.…”

“…Electric field induced birefringence and circular dichroism studies indicate that the bases in poly(A) become unstacked, at least partially, upon binding E. coli SSB protein (Kuil et al, 1990). On the other hand, since the bases within free dT(pT)" or dC(pC)" are not stacked significantly, similar base unstacking should not occur upon SSB binding to dT(pT)" or dC(pC)".…”

“…The base-unstacking model is based on the fact that the bases within dA(pA)" undergo significant unstacking with increasing temperature (Leng & Felsenfeld, 1966;Riley et al, 1966;Poland et al, 1966;Ts'o et al, 1966;Vournakis et al, 1967), whereas dT(pT)" and dC(pC)" show negligible base stacking (Felsenfeld & Miles, 1967;Riley et al, 1966;Ts'o et al, 1966). However, when bound to SSB protein, the bases in dA(pA)" become unstacked, at least partially (Kuil et al, 1990). In this model, the DNA exists in equilibrium among a continuum of conformational states differing in the extent of base stacking; however, the SSB protein is assumed to bind only to the unstacked state of the DNA, Au.…”

Protein Interactions in Genome Maintenance as Novel Antibacterial Targets

“…The first is that SSB/protein interactions could have important, but previously unrecognized, roles protein translation. Indeed, SSBs from E. coli and bacteriophage are known to bind single-stranded RNA in a manner that influences translation [45]–[47] [48], [49]. Moreover, proteomic studies have shown that SSB from both E. coli and B. subtilis associate with ribosomal subunits [43], [50], although, due to the prevalence of ribosomal proteins as common contaminants in proteome-wide studies, the possibility that these are bona fide interactions has not be pursued further.…”

Cooperative binding of polyamines induces the Escherichia coli single-strand binding protein-DNA binding mode transitions

“…velocity (Lohman & Overman, 1985; Lohman et al, 1986; Bujalowski & Lohman, 1986;Bujalowski et al, 1988), and circular dichroism (Kuil et al, 1990). In complexes with poly(dT) at 25 °C, pH 8.1, the SSB tetramer can form three of these modes with = 35, 56, or 65 nucleotides per tetramer.…”