Budding Yeast Rad9 Is an ATP-Dependent Rad53 Activating Machine Christopher S Gilbert, Catherine M Green, Noel F Lowndes  Molecular Cell  Volume 8, Issue 1, Pages 129-136 (July 2001) DOI: 10.1016/S1097-2765(01)00267-2

Figure 1 Rad9 Hydrodynamic Analyses (A) Size exclusion chromatographic analyses of Rad9 complexes. Fractions from a Superose 6 column loaded with crude cell extracts from wild-type cells were Western blotted and probed with anti-Rad9 serum. (B) Sedimentation analyses of Rad9 complexes. Fractions from glycerol velocity sedimentation gradients were Western blotted and probed with anti-Rad9 serum. −UV and +UV indicate either mock treated or UV irradiated. The relative migration positions of hyperphosphorylated (Rad9-P) and hypophosphorylated (Rad9) Rad9 are indicated. Letters below the panels indicate the position of standard proteins (masses, stokes radii, and sedimentation coefficients, respectively are listed). T, thyroglobulin (670 kDa, 8.5 nm, 18 × 10−13 s); F, ferritin (440 kDa, 6.1 nm, 65 × 10−13 s); Ca, catalase (232 kDa, 5.2 nm, 11.3 × 10−13 s); A, aldolase (158 kDa, 4.8 nm, 7.4 × 10−13 s); B, bovine serum albumin (67 kDa, 3.6 nm, 4.3 × 10−13 s); O, ovalbumin (44 kDa, 3.1 nm, 3.7 × 10−13 s); Ch, chymotrypsinogen (25 kDa, 2.1 nm, sedimentation coefficient not known); DB, dextran blue (2000 kDa) Molecular Cell 2001 8, 129-136DOI: (10.1016/S1097-2765(01)00267-2)

Figure 2 Expression of Rad53 in E. coli Results in its Autophosphorylation (A) Western blot of samples from a time course of histidine-tagged Rad53 (H-Rad53) induction in E. coli, using serum JDI48. The lower panel is a longer exposure of a portion of the top panel, shown to reveal hypophosphorylated H-Rad53 present in the 5 min sample and the relative migration positions of phosphorylated (+, after UV irradiation, 50 J/m2) and hypophosphorylated (−) Rad53 from wild-type yeast cells loaded as controls. (B) A Coomassie-stained 10% SDS-PAGE gel of hyperphosphorylated recombinant H-Rad53 purified from E. coli (lane 1) and the same material treated with either 4 (lane 2) or 0.4 (lane 3) units of λ protein phosphatase per μg of recombinant protein Molecular Cell 2001 8, 129-136DOI: (10.1016/S1097-2765(01)00267-2)

Figure 6 A Model for Rad9 Function The ≥850 kDa hypophosphorylated Rad9 complex may associate either directly or indirectly with damaged DNA. Alternatively, it may simply be a latent Rad9 complex that must be activated by Mec1-dependent phosphorylation. Shown in (A) is the possibility that the ≥850 kDa Rad9 complex can indirectly associate with DNA lesions in the context of repair proteins and Mec1. In principal, either the ≥850 kDa Rad9 complex or Mec1 could bind first. Alternatively, the ≥850 kDa Rad9 complex could act as a Mec1 cofactor, facilitating Mec1 recruitment to DNA lesions. Regardless of the exact order of function, the model suggests that interaction between the chromatin-bound ≥850 kDa Rad9 complex and Mec1 (in the context of a DNA lesion undergoing processing) must be brief, as Mec1-dependent hyperphosphorylation of Rad9 results in its release as the 560 kDa soluble complex. It is not known whether the conformational change and loss of mass observed for the ≥850 kDa Rad9 complex is a consequence of, or a prerequisite for, hyperphosphorylation of Rad9, although the copurification of Ssa1 and/or Ssa2 with both soluble forms of the Rad9 complex suggest that chaperone activity is required (C.S. Gilbert et al., submitted). However, this remodeling process involves breakage of salt-sensitive Rad9-Rad9 interactions and formation of salt-resistant interactions dependent on the Rad9 BRCT domain (Soulier and Lowndes, 1999). Hyperphosphorylation of Rad9 results in recruitment of the Rad53 protein kinase to Mec1-generated phospho residues on Rad9 to form the soluble 560 kDa complex shown in (B). Rad53 molecules are thereby brought into close proximity, facilitating in trans autophosphorylation Rad53. Activated Rad53 is released in an active process requiring ATP hydrolysis. The released Rad53 (C) specifically targets substrates required for checkpoint pathway-regulated cell cycle arrest, transcriptional induction of the DNA damage regulon, and efficient DNA repair. Note that the stoichiometries of the Rad9 complexes indicated in the model are just one of several possibilities consistent with the molecular mass estimates. The suggested stoichiometry of the ≥850 kDa Rad9 complex is based on an assumption that this complex is a dimer of the core (that is, excluding Rad53) 560 kDa complex Molecular Cell 2001 8, 129-136DOI: (10.1016/S1097-2765(01)00267-2)

Figure 3 Hyperphosphorylated Rad9 Functions in Catalysis of Rad53 Autophosphorylation (A) Rad53 release assay using partially purified HH-Rad9 complexes obtained from UV-irradiated (50 J/m2) cells. The amount of Rad9 and Rad53 remaining bound to 12CA5 agarose beads (Beads) or released into the supernatant (Sup) after incubation with ATP (+), ATP-γS (γS), or mock treatment without any nucleotide (−) was determined by Western blotting. The release assays were carried out either in the presence of potassium (K+) or sodium (Na+) salts. The load (L) material prior to binding to the beads and material that did not bind to the beads (FT) are indicated. Lane 1 is loaded with extract from nonirradiated wild-type yeast cells to show the relative migration position of hypophosphorylated Rad9 and Rad53, as indicated. As the anti-Rad53 polyclonal serum used (JDI48) does not detect nonphosphorylated Rad53 efficiently, a longer exposure of lane 1 for the Rad53 Western blot is shown. (B) The hyperphosphorylated Rad9 complex can catalyze the phosphorylation of recombinant Rad53 (H-Rad53). A Rad53 Western blot of 10 μg crude extract from nonirradiated wild-type cells (lane 1), 10 μg crude extract from UV-irradiated cells (lane 2), or 2.5 ng dephosphorylated H-Rad53 (lane 3). Rad9 beads were prepared from UV-irradiated wild-type cells (Rad9-P) or UV-irradiated rad9Δ cells (Δ). These were incubated in the presence of 50 ng dephosphorylated recombinant Rad53 (H-Rad53) and ATP. After the incubation, the H-Rad53 was removed from any endogenous Rad53 by Ni2+-NTA chromatography and analyzed by Western blotting using polyclonal serum, JDI48 (lanes 4–6). One milligram of extract was bound to the 12CA5 beads for the reactions shown in lanes 4 and 6, whereas 0.5 mg of extract was used for lane 5. Equivalent amounts of H-Rad53 were run on SDS-PAGE gels for the Western blot Molecular Cell 2001 8, 129-136DOI: (10.1016/S1097-2765(01)00267-2)

Figure 4 Release of Rad53 from the Rad9 Complex Requires Rad53 Kinase Activity Twenty micrograms of crude extract from undamaged wild-type (lane 1), UV-irradiated wild-type (lane 2), and UV-irradiated RAD53K227A (lane 3) cells were loaded to locate the relative migration positions of Rad9 and Rad53 phospho forms. Rad53 release assays were performed with UV-irradiated wild-type extract (WT, lanes 4 and 5) and UV-irradiated RAD53K227A extract (KD, lanes 6 and 7). For each release assay, the supernatant (S) and beads (B) obtained after the ATP incubation are indicated Molecular Cell 2001 8, 129-136DOI: (10.1016/S1097-2765(01)00267-2)

Figure 5 In Vitro Complementation of the Rad53 Release Assay A Rad53 Western blot of supernatants from Rad53 release assays performed with partially and highly purified HH-Rad9 complexes from UV-irradiated wild-type cells. Lanes 1 and 2: supernatants from partially purified (12CA5 affinity) HH-Rad9 complexes either incubated without (−) or with (+) ATP as indicated. Lane 3: supernatant from the highly purified HH-Rad9 complex (12CA5 plus Ni2+-NTA) treated with ATP. Lanes 4 and 5, as for lane 3, except the reactions were also complemented in vitro with 1 μg crude cell extract from wild-type or mec1-1 tel1Δ cells, respectively. Lanes 6 and 7: control for the Rad53 signal from 1 μg wild-type or mec1-1tel1Δ crude cell extracts, respectively Molecular Cell 2001 8, 129-136DOI: (10.1016/S1097-2765(01)00267-2)