Characterization of the DNA-binding properties of the origin-binding domain of simian virus 40 large T antigen by fluorescence anisotropy

Abstract

The affinity of the origin-binding domain (OBD) of simian virus 40 large T antigen for its cognate origin was measured at equilibrium using a DNA binding assay based on fluorescence anisotropy. At a near-physiological concentration of salt, the affinities of the OBD for site II and the core origin were 31 and 50 nM, respectively. Binding to any of the four 5'-GAGGC-3' binding sites in site II was only slightly weaker, between 57 and 150 nM. Although the OBD was shown previously to assemble as a dimer on two binding sites spaced by 7 bp, we found that increasing the distance between both binding sites by 1 to 3 bp had little effect on affinity. Similar results were obtained for full-length T antigen in absence of nucleotide. Addition of ADP-Mg, which promotes hexamerization of T antigen, greatly increased the affinity of full-length T antigen for the core origin and for nonspecific DNA. The implications of these findings for the assembly of T antigen at the origin and its transition to a non-specific DNA helicase are discussed.

FIG. 1.

Purified proteins used in this study. (A) Schematic diagram of the 708-amino-acid SV40 large T antigen. The position of the OBD, located between amino acids 131 and 260, is indicated relative that of other functional domains including the J domain (J), the ATPase domain and the zinc finger domain (Zn). Regions of the protein involved in binding to the cell cycle regulatory protein Rb and p53 are indicated as black bars. (B) Sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis of purified proteins stained with Coomassie blue. Aliquots (3 μg) of full-length large T antigen, of polyhistidine-tagged OBD (His-OBD), and of the corresponding GST fusion protein (GST-His-OBD) were analyzed. Concentrations of the His-OBD and Gst-His-OBD proteins were estimated by absorbance at 280 nm in 6 M guanidine hydrochloride using the following molar extinction coefficients: His-OBD = 9,320 M⁻¹cm⁻¹; GST-His-OBD = 50,480 M⁻¹cm⁻¹. The concentration of full-length T antigen was determined by Bradford analysis. (C and D) Gel filtration profiles of His-OBD (C) and Gst-His-OBD (D) proteins. His-OBD (75 μg) and GST-His-OBD (150 μg) were chromatographed on a Superdex 75 PC 3.2/3.0 and a Superdex 200 PC 3.2/3.0 gel filtration column, respectively, using the SMART system (Pharmacia) in a solution containing 25 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol. Molecular mass standards in (C) were blue dextran (2,000 kDa), albumin (67.0 kDa), ovalbumin (43.0 kDa), chymotrypsinogen A (25.0 kDa), and RNase A (13.7 kDa). These standards were chromatographed in two groups to increase peak resolution; both elution profiles are superimposed in the figure. Molecular mass standards in panel D were thyroglobulin (670 kDa), gamma globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B₁₂ (1.35 kDa). The positions of each standard and of the void volume (V₀) are indicated by black triangles. The molecular masses of the His-OBD and of GST-His-OBD proteins calculated from their retention times relative to those of the standards are 15.8 and 92 kDa, respectively. These values are consistent with the His-OBD and of GST-His-OBD proteins being primarily monomeric and dimeric, respectively (molecular mass of a monomer calculated from the primary amino acid sequence is 16.0 kDa for His-OBD and 42 kDa for Gst-His-OBD).

FIG. 2.

DNA duplexes used in this study. (A) Fluorescent probes and analogous competitor oligonucleotides. The sequences of the fluorescein (F)-labeled strand of the probe containing a single T antigen-binding site (TBS) and of the control probe are indicated. Similarly, the top strand of competitor oligonucleotides containing one, two, or no TBS is indicated. TBS are boxed in black. (B) Oligonucleotides derived form the SV40 origin. The sequence of the 64-bp double-stranded core origin is indicated. The AT-rich and early palindrome regions are boxed. The positions of site II and of the four pentanucleotide TBS are indicated. The bottom of the figure describes the sequence of the top strand of competitor oligonucleotides derived from the 31-bp site II or from the 64-bp core origin. Functional (not mutated) TBS are boxed in black and their numbers are specified in the nomenclature of each oligonucleotide. Site II oligonucleotides in which the spacer region between TBS 1 and 3 has been increased by 1, 2, or 3 bp are referred to as +1, +2, and +3, respectively.

FIG. 3.

DNA-binding activity of the large T-antigen OBD and of full-length large T antigen detected by fluorescence anisotropy. Binding isotherms were generated with purified His-OBD (A) or GST-His-OBD (B) at room temperature in binding buffer (50 mM Tris [pH 7.6], 0.005% NP-40, 1 mM EDTA, 1 mM dithiothreitol) containing 100 mM NaCl and a 15 nM concentration of either a one-TBS-containing probe (circles) or a control probe lacking any TBS (triangles). Binding isotherms generated with large T antigen (C and D) were obtained at room temperature in binding buffer containing the indicated concentration of NaCl and either a one-TBS-containing probe (C) or a control probe lacking any TBS (D).

FIG. 4.

Model for the assembly of SV40 T antigen at the origin and comparison with the assembly of the related papillomavirus E1. (A) Assembly of the T-antigen OBD on site II. The DNA is represented in gray. The four pentanucleotides TBS are indicated by arrows and numbered according to Fig. 2. The figure shows the stepwise, independent binding of four OBD (colored in black) onto the four TBS in site II. Assembly is shown to proceed stepwise starting with binding of the OBD to the highest affinity site (site 3) and ending with binding to the weakest site (site 2). (B) Assembly of the papillomavirus E1 DBD at its cognate origin. The four E1 binding sites found in many papillomavirus origins (4) are indicated by arrows as two overlapping pairs of inverted repeats. Assembly is shown to begin by the binding of a monomer of the E1 DBD (black box) to the highest-affinity binding site, site 4 for HPV11 (26). The affinity of a monomer for the origin is weak (K_i = 850 nM for the HPV11 E1 DBD at 100 mM NaCl [26]), but its binding is increased approximately 10-fold upon dimerization (for a dimer of the HPV11 E1 DBD K_i = 80 nM at 100 mM NaCl [26]). Assembly is shown to proceed via the cooperative binding of two E1 DBD on sites 2 and 4 to form an initial stable dimer. This is followed by the assembly of a second dimer on the weaker affinity sites 1 and 3 (26). A recent crystal structure of the tetrameric E1 DBD-DNA complex revealed that both dimers do not interact with each other significantly (11). The low affinity and specificity of E1 for its target site are such that, in vivo, dimerization of full-length E1 probably occurs only if the protein is recruited to the origin by E2, a transcription/replication factor with high affinity for the origin.