Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding

Abstract

The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded onto chromatin by its loader complex, Rad17-RFC, following DNA damage. The 120-amino acid (aa) stretch of the Rad9 C terminus (C-tail) is unstructured and projects from the core ring structure (CRS). Recent studies showed that 9-1-1 and CRS bind DNA independently of Rad17-RFC. The DNA-binding affinity of mutant 9(ΔC)-1-1, which lacked the Rad9 C-tail, was much higher than that of wild-type 9-1-1, suggesting that 9-1-1 has intrinsic DNA binding activity that manifests in the absence of the C-tail. C-tail added in trans interacted with CRS and prevented it from binding to DNA. We narrowed down the amino acid sequence in the C-tail necessary for CRS binding to a 15-aa stretch harboring two conserved consecutive phenylalanine residues. We prepared 9-1-1 mutants containing the variant C-tail deficient for CRS binding, and we demonstrated that the mutant form restored DNA binding as efficiently as 9(ΔC)-1-1. Furthermore, we mapped the sequence necessary for TopBP1 binding within the same 15-aa stretch, demonstrating that TopBP1 and CRS share the same binding region in the C-tail. Indeed, we observed their competitive binding to the C-tail with purified proteins. The importance of interaction between 9-1-1 and TopBP1 for DNA damage signaling suggests that the competitive interactions of TopBP1 and CRS with the C-tail will be crucial for the activation mechanism.

Keywords: DNA damage response; DNA-protein interaction; checkpoint control; clamp protein; protein phosphorylation; protein/protein interaction.

FIGURE 1.

DNA binding of 9^ΔC-1-1 analyzed by EMSA. Autoradiographs of EMSA with 9-1-1 or 9^ΔC-1-1 under various conditions are shown. A, increasing amounts of 9-1-1 or 9^ΔC-1-1 (0, 0.06, 0.17, 0.5, 1.5, 3, and 6 pmol each) were incubated with 5′ end DNA. B, 9^ΔC-1-1 (2 pmol) was incubated with 5′ end DNA, with or without 250 ng of anti-FLAG monoclonal antibody. A super-shifted band appeared in the presence of the antibody. C, 9^ΔC-1-1 (0, 0.75, or 1.5 pmol) was incubated with the 5′ end, 3′ end, ssDNA, and dsDNA. D, 9^ΔC-1-1 (0, 1.5, or 3 pmol) was incubated with ssDNA or dsDNA with lengths ranging from 30- to 90-mer. E, 9^ΔC-1-1 (0, 2.5, and 5 pmol) was incubated with various 49-mer DNAs (ss, ds, 5′ end, 3′ end, and fork). It should be noted that differences among experiments in DNA-binding efficiencies were the result of different specific activities of 9^ΔC-1-1 preparations. Intensities of the shifted bands in A, C, D, and E were quantified, and their ratios (%) relative to total DNA are graphed on the right side or below. Values represent the means of three independent experiments, and error bars indicate S.E. A, inset is K_d of 9^ΔC-1-1, which was estimated as 2.2 × 10⁻⁷ from the quantified shifted bands, although the values of the shifted bands of 9-1-1 were too low to estimate the precise K_d value from this study.

FIGURE 2.

Binding of C-tail with 9-1-1 and 9^ΔC-1-1. A and B, glutathione-Sepharose beads pre-bound with GST only or GST/FLAG-tagged C-tails (from E. coli cells in A, and EC and HF in B from E. coli and insect cells, respectively) were mixed with 10 pmol each of purified 9-1-1 or 9^ΔC-1-1 at 4 °C for 1 h. After washing the beads, input proteins (1%) and bound proteins (20%) were analyzed by immunoblotting using the indicated antibodies for 9-1-1 subunits (upper panel), and staining with Coomassie Brilliant Blue (A) or Ponceau S (B) for C-tail (lower panels). Asterisks indicate degraded C-tail fragments.

FIGURE 3.

Analyses of amino acid sequence of C-tail necessary for its binding to 9^ΔC-1-1. A, schematic illustration of human C-tail and its deletions used in this study. C-tail, C-N, and C-C contain aa 272–391, aa 272–332, and aa 333–391, respectively. Constructs Δ336, Δ341, Δ351^d5, Δ351, Δ356, Δ361, and Δ371 harbor internal deletions of aa 336–345, aa 341–350, aa 351–355, aa 351–360, aa 356–365, aa 361–370, and aa 371–380 from C-C, respectively; C-tail^Δ351d5 and C-tail^Δ351 are the same deletions as Δ351^d5 and Δ351, respectively, from the C-tail. C-tail^FFAA harbors Ala substitutions at both Phe-365 and Phe-366, which were expressed as fusions with GST/FLAG tags at their N-terminal portion. Below is the sequence of the C-tail, including the 15-aa stretch (aa 351–375) required for interaction with CRS. The NLS and FF motif (Phe-365/Phe-366) are boxed in white and black, respectively. B, glutathione-Sepharose beads pre-bound with GST only (mock), GST/FLAG-tagged C-tail (C-tail), C-tail mutant (C-tail^FFAA), or its deletions (C-N, C-C, Δ336, Δ341, Δ351^d5, Δ351, Δ356, Δ361, Δ371, C-tail^Δ351d5, and C-tail^Δ351) were mixed with 10 pmol alone (left panel) or 5 and 10 pmol (+, and ++, respectively) (right panel) of purified 9^ΔC-1-1, and incubated at 4 °C for 1 h. After washing the beads, 50 fmol (left panel) and 100 fmol (right panel) of purified 9^ΔC-1-1 and 10% (left panel) or 20% (right panel) of the bound fractions were analyzed by immunoblotting using anti-Rad1 antibody (upper panels) and staining with Ponceau S for the C-tail and its deletions (lower panels). Asterisks indicate degraded C-tail fragments.

FIGURE 4.

Effects of intramolecular binding on DNA binding by 9-1-1. A, increasing amounts of C-tail or C-tail^FFAA (2.5, 5, 10, or 20 pmol) were added to a DNA-binding assay containing 9^ΔC-1-1 (2 pmol) and ssDNA (90-mer, 5 fmol) and analyzed as in Fig. 1 (left panel). The ratios of bound DNA (% of total DNA) obtained as the means of two independent experiments as in Fig. 1D are plotted in the right graph; error bars indicate S.E. B, increasing amounts of 9-1-1 and 9-1-1 mutant (9^ΔC-1-1, 9^Δ351d5-1-1, 9^Δ351-1-1) (1, 2, or 4 pmol each) were incubated with ssDNA, analyzed (left panel), and quantitated (right panel) as in A.

FIGURE 5.

Analyses of the interaction of TopBP1 with C-tail, its Ala substitution mutant, and its deletions. A, C-tail fragments used for this analysis as indicated in Fig. 3A, except for C-C^2A, which contains Ala substitutions at both Ser-341 and Ser-387. B, glutathione-Sepharose beads pre-bound with GST only (mock), GST/FLAG-tagged C-tail (C-tail), and its derivatives were prepared as Fig. 3, except for treatment with CK2 for 1 h at 30 °C, followed by three washes. A portion (20%) of the bound fractions was used for Coomassie Brilliant Blue (CBB) staining (upper left panel), and 0.5% was used for immunoblotting with the indicated antibodies (remaining panels). C and D, prepared beads as B with (C and D) or without (C) CK2 treatment were mixed with 5 or 10 μl of lysates of insect cells expressing TopBP1; 1 μl of the lysates and 25% of the bound fractions were analyzed by Coomassie Brilliant Blue staining. Asterisks indicate degraded C-tail fragments.

FIGURE 6.

TopBP1 and CRS compete each other for C-tail. A, anti-FLAG beads were pre-bound with 10 pmol of purified FLAG-tagged 9^ΔC-1-1 and incubated with GST-tagged C-tail with or without CK2 phosphorylation (P-C-tail or C-tail) at 4 °C for 1 h. After washing the beads, 0, 3, or 5 pmol (−, +, or ++, respectively) of purified TopBP1 were added and further incubated for 1 h at 4 °C. One percent of the input fractions and 30% of the bound fractions were analyzed by immunoblotting using indicated antibodies. B, band intensities of C-tail in the bound fractions were divided by that of Rad9^ΔC and the values relative to TopBP1 minus experiments as 100% were plotted as means ± S.E. for four independent experiments.