Examination of a putative interaction motif and the predicted coiled region of Rad24 and their role in the Rfa1-Rad24 interaction and cellular response to DNA damage.

[A] Sequence display of Rad24 C-terminal region and the residues predicted to form a coiled structure. Jpred3 prediction probabilities are denoted below each residue, where the single digit represents the probability on a scale of 0-10 (e.g., 9 = 90%), rounded to the nearest digit. Probability approaching 100% is denoted by an asterisk. COILS examined the region in windows of 14, 21, and 28 amino acid residues (denoted for each). The denoted coil (C) from Jpred3 was predicted using Lupas [39] and was only predicted for a window of 21 aa. The predicted coil region is denoted by black highlighting. Junction represents the region that was deleted in ΔC2 (2) or ΔC3 (3). Above the amino acid sequence for Rad24 are “interaction motifs” predicted by Oakley and Patrick based on known interaction regions for these human proteins and human Rpa1 [1]. [B] Mutation of a predicted Rfa1 interaction motif does not disrupt interaction. The aspartic acid residues (D593 and D594) in the motif DDLE were mutated to alanines in the Rad24 C-terminal peptide, and interaction with Rfa1 was assessed via two-hybrid analysis as in Fig. 1C. Also examined was a mutant form where two glutamic acid residues (E598 and E599) in the predicted coil were mutated to alanines. Both mutant forms display growth on SG-HTUL and blue color on SG-HTU+X-gal that is as robust as the Rfa1-Rad24-ΔN interaction. [C] Deletion of the coil partially disrupts interaction. The predicted coil region was deleted and interaction between Rfa1 and the Rad24 C-terminus was assessed by replica plating as in [B]. Blue color on SG-HTU+X-gal is reduced compared to that for Rfa1-Rad24-ΔN. [D] Physiological consequences of deleting or mutating the Rad24 C-terminal region. DNA damage spot assays were performed, and the entire range of testing is shown in S4 and S5 Figs. rad24Δ cells (denoted vertically on left) containing an empty vector (EV) are sensitive to camptothecin (CPT) and ultraviolet radiation (UV). This can be complemented by a plasmid expressing the wild-type RAD24 gene from its endogenous promoter. The following mutations were made in this plasmid and examined for sensitivity to CPT or HU: DD→RR = charge reversal mutant for D593 and D594; Δcoil = deletion of amino acids (aa) 575–601 containing the predicted coil; ΔC2 or ΔC3 = deletions of aa 528–594 or 595–659, respectively. WT denotes an isogenic strain containing the wild-type copy of RAD24 in the chromosome.