Binding mode transitions of Escherichia coli single strand binding protein-single-stranded DNA complexes. Cation, anion, pH, and binding density effects.

W Bujalowski,L B Overman,T M Lohman

doi:10.1016/s0021-9258(18)68829-5

W Bujalowski, L B Overman + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)68829-5

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Journal: The Journal of biological chemistry	Publication Date: Apr 1, 1988
Citations: 149	License type: cc-by

Affiliation: Texas A&M University

Abstract

We have extended our investigations of the multiple binding modes that form between the Escherichia coli single strand binding (SSB) protein and single-stranded DNA (Lohman, T. M. & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W. & Lohman, T. M. (1986) Biochemistry 25, 7799-7802) by examining the effects of anions, pH, BaCl2, and protein binding density on the transitions among these binding modes. "Reverse" titrations that monitor the quenching of the intrinsic tryptophan fluorescence of the SSB protein upon addition of poly(dT) have been used to measure the apparent site size of the complex at 25 degrees C in pH 8.1 and 6.9 as a function of NaF, NaCl, NaBr, and MgCl2 concentrations. Under all conditions in which "reverse" titrations were performed, we observe three distinct binding modes with site sizes of 35 +/- 2, 56 +/- 3, and 65 +/- 3 nucleotides/SSB tetramer; however, the transitions among the three binding modes are strongly dependent upon both the cation and anion valence, type, and concentration as well as the pH. A net uptake of both cations and anions accompanies the transitions from the (SSB)35 to the (SSB)56 binding mode at pH 6.9, whereas at pH 8.1 this transition is anion-independent, and only a net uptake of cations occurs. The transition from the (SSB)56 to the (SSB)65 binding mode is dependent upon both cations and anions at both pH 6.9 and 8.1 (25 degrees C), and a net uptake of both cations and anions accompanies this transition. We have also examined the transitions by monitoring the change in the sedimentation coefficient of the SSB protein-poly(dT) complex as a function of MgCl2 concentration (20 degrees C, pH 8.1) and observe an increase in s20,w, which coincides with the increase in apparent site size of the complex, as measured by fluorescence titrations. The frictional coefficient of the complex decreases by a factor of two in progressing from the (SSB)35 to the (SSB)65 binding mode, indicating a progressive compaction of the complex throughout the transition. The transition between the (SSB)35 and the (SSB)56 complex is dependent on the protein binding density, with the lower site size (SSB)35 complex favored at higher binding density. These results indicate that the transitions among the various SSB protein-single-stranded DNA binding modes are complex processes that depend on a number of solution variables that are thermodynamically linked.(ABSTRACT TRUNCATED AT 400 WORDS)

Highlights

From th.e Departments of $Biochemistry and Biophysics and of Whemistry, Texas A&M University, College Station, Texas 77843
We have examined the transitions by monitoring the change in thsedimentation coefficient of the structures arelikely to be the (SSB) protein-poly(dT) complex as a function of MgCl, concentration (20O C, pH 8.1)and observe an increase in S Z O, ~, which coincides with the increase in apparent sitesize of the complex, as measured by flucase of the Escherichia coli SSB protein, several of these interactions have been identified in vitro (Sigal et al, 1972; Molineux et al, 1974; Molineuxand Gefter, 1975; Low et al, 1982), andothers have been inferred from genetic studies (Tessman and Peterson, 1982).The E. coli SSB protein stimulates the DNA strand exchange activity of the recA protein under some conditions (Cox and Lehman, 1981,1982; Griffth et al, 1984)
The midpoints of the (SSB),6 to (SSB), transition occur at approximately 80, 160, and 600 mM for Cation-specificEffectson the SSB Protein-Poly(dT) Binding Mode Transitions-In previous experiments with poly(dT), we have shown that the transitionsamong the various E. coli SSB protein-ssDNA binding modes can be induced at much lower concentrations ofMgCl, than NaCl (Bujalowski and Lohman, 1986)