Supplemental Material

This article contains the following supporting material:

• Figure 1 - Phosphorylation of Rad1 on T5 is ATRIP-dependent and Claspin-independent. (A) ATRIP is required for phosphorylation of Rad1 on T5. 10 μL egg cytosol was mock-depleted (ΔlgG) or immunodepleted with anti-ATRIP (ΔATRIP) antibodies and incubated with exoIII-treated plasmid (2.5 ng) for 30 minutes. Rad1, P-T5 Rad1, and ATRIP were visualized by immunoblotting. (B) Rad1 phosphorylation on T5 is Claspin-independent. Extract was mock-depleted (ΔlgG) or immunodepleted with anti-Claspin (ΔClaspin) antibodies and incubated with exoIII-plasmid as in A. Rad1, P-T5 Rad1, and Claspin were analyzed by immunoblotting.

• Figure 2 - Rad9 domain mapping and independence of Hus1 phosphorylation from C-terminus of Rad9. (A) Mapping of the minimal Hus1/Rad1 interaction domain in Rad9. Untagged full length-Rad9 (UT-377), FLAG-Rad9 full length (FLAG-377) or C-terminal deletions of Rad9 (1-270, FLAG-270; 1-280, FLAG-280; 1-290, FLAG-290; 1-305, FLAG-305) cDNAs were co-in vitro translated with Hus1 and Rad1 in the presence of ³⁵S-methionine and incubated with anti-FLAG agarose beads. Input proteins (top panel) and immunoprecipitated proteins (bottom panel) were visualized by autoradiography. (B) Hus1 is phosphorylated in the absence of the C-terminus of Rad9. In vitro translated ³⁵S-labeled Hus1 was incubated with unlabeled Rad1 and ³⁵S-labeled full-length Rad9, (1-305)Rad9, or unlabeled (1-280)Rad9 to form RHR complexes as described in Figure 7. 2 μL of labeled RHR complexes were added to 10 μL egg cytosol and treated with buffer or exoIII-plasmid (5 ng). After 30 minutes, samples were analyzed by SDS-PAGE and autoradiography. (C) Rad1 and Hus1 are not phosphorylated in the absence of the other members of the RHR complex. In vitro-translated, ³⁵S-labeled Rad1 or Hus1 were individually incubated in 10 μL egg cytosol immunodepleted of endogenous RHR complex in the absence or presence of exoIII-treated plasmid (5 ng). After 30 minutes, Rad1 and Hus1 were visualized by autoradiography.