Study of the binding of single-stranded DNA-binding protein to DNA and poly(rA) using electric field induced birefringence and circular dichroism spectroscopy.
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.
Kuil
M E
ME
Department of Physics and Astronomy, Free University de Boelelaan, Amsterdam, The Netherlands.
Holmlund
K
K
Vlaanderen
C A
CA
van Grondelle
R
R
D001426:Bacterial Proteins / Q000378:metabolism*; D001718:Birefringence; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D011061:Poly A / Q000378:metabolism*
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011061:Poly A; D004247:DNA