Mms1 and Mms22 regions involved in complex formation. A, identification of the Cul8- and Mms22-binding domains in Mms1. Baculoviruses expressing Myc-Cul8, His₆HA-Mms22, and full-length or truncated mutants of FLAG-Mms1 were co-transfected into Sf21 cells. The cells were lysed, and the tagged proteins were immunoprecipitated and then analyzed by immunoblotting. The left panel demonstrates schematic diagrams of Mms1 deletion mutants. The asterisk indicates a cross-reacting band. B, identification of the Mms1- and Esc4-binding domains in Mms22. Baculoviruses expressing His₆HA-Mms22, HSV-Esc4, and full-length or truncated mutants of FLAG-Mms1 were transfected separately into Sf21 cells. The cells were lysed, and the lysates were mixed to equalize the amounts of the Mms22 proteins in the mixtures. The proteins were then immunoprecipitated from the mixtures and analyzed by immunoblotting. The left panel demonstrates schematic diagrams of Mms22 deletion mutants. IP, immunoprecipitation; WT, wild type.

Interaction of Cul8-Mms1 with Esc2 or Orc5. A, Esc2 immunoprecipitated with Cul8 and Mms1 but not with Mms22. FLAG-Esc2 was expressed from a single-copy plasmid under the control of the ADH1 promoter. Endogenous Cul8-HA, Mms1-HA, or Mms22-HA was immunoprecipitated with anti-HA, and then any FLAG-Esc2 that had co-immunoprecipitated was identified by immunoblotting with anti-FLAG. The asterisk indicates a cross-reacting band. B, co-immunoprecipitation of Cul8-Myc or Mms1-HA with Esc2. FLAG-Esc2 was expressed from a single-copy plasmid under the control of the ADH1 promoter. FLAG-Esc2 was immunoprecipitated with anti-FLAG, and then endogenous Cul8-Myc and Mms1-HA that had co-immunoprecipitated were identified by immunoblotting. C, co-immunoprecipitation of Cul8-Myc or Mms1-HA with Orc5. The experiments were performed as in C, except that Orc5-FLAG was used instead of Esc2-FLAG. IP, immunoprecipitation.

Interaction between Ctf4 and Cul8. A, yeast strains, with their genotypes indicated to the left of the figure, were grown to an A₆₀₀ of 1.0 and serially diluted in 10-fold increments, and then 4 μl of the serial dilutions was spotted onto YPD plates, containing 50 mm hydroxyurea, 0.004% MMS, or 2 μm camptothecin. The plates were incubated at 30 °C for the indicated number of days. B, yeast strains were cultured in YPD, then treated with or without MMS for 1.5 h. Open circles, wild type (WT); closed circles, cul8Δ; open triangles, ctf4Δ; closed triangles, cul8Δctf4Δ; open squares, rad54Δ; closed squares, cul8Δrad54Δ. The cells were plated onto YPD plates. Two days later, the colony numbers were counted. C and D, the spot assay was performed in the same manner as the experiments of Fig. 6A.

Cul8-Mms1-Mms22-Esc4 complex formation. A, yeast cell extracts were prepared from the Cul8–3FLAG-expressing strain. Proteins that bound to Cul8 in vivo were isolated by immunoprecipitation with anti-FLAG, resolved by SDS-PAGE, and then stained with Coomassie Brilliant Blue. B, baculoviruses expressing Myc-Cul8, FLAG-Mms1, and His₆-HA-Mms22 were co-transfected into Sf21 cells. The cells were lysed, and the proteins were immunoprecipitated with antibodies to the three tags. Immunoprecipitates were analyzed by immunoblotting. α means anti-. C, yeast strains YSI25 (containing Cul8-Myc, Mms1-HA, and Mms22-Pk) and YSI38 (containing Cul8-Myc, mms1Δ, and Mms22-Pk) were cultured in YPD. The proteins in extracts were immunoprecipitated with the antibodies used for the experiments of Fig. 1B, and immunoprecipitates were analyzed as in B. D, baculoviruses expressing Myc-Cul8, FLAG-Mms1, His₆HA-Mms22, and HSV-Esc4 were co-transfected into Sf21 cells. Immunoprecipitation was performed, and the immunoprecipitates were analyzed in the same manner as the experiments of Fig. 1B. E, yeast strain YSI29, for which its endogenous Cul8, Mms1, Mms22, and Esc4 were epitope-tagged as indicated in the figure, was cultured in YPD. Immunoprecipitation was performed, and the immunoprecipitates were analyzed as for the experiments shown in Fig. 1B. F, yeast strains, YSI29 (for which its endogenous Cul8, Mms1, Mms22, and Esc4 were epitope-tagged as indicated in the figure) and YSI38 and YSI34 (which were identical to YSI29 except that MMS1 or MMS22 was deleted, respectively) were cultured in YPD. Immunoprecipitation was performed, and the immunoprecipitates were analyzed in the same manner as the experiments in B. IP, immunoprecipitation; WT, wild type.

Identification of Esc2, Orc5, and Ctf4 as components of a Cul8 complex. A, two-hybrid experiments. The yeast strain, L40, was transformed with bait (BD) plasmids and prey (AD) plasmids. Yeast strains were plated onto synthetic defined plates, with or without histidine, or various amount of 3-amino-1,2,4-triazole (3-AT). Because LexA-Mms22, to some extent, self-activated HIS3, at least 10 mm of 3-AT was required to suppress the background level of expression. B, co-immunoprecipitation of the Cul8 complex with Ctf4, Esc2, or Orc5. FLAG-Ctf4, FLAG-Esc2, or FLAG-Orc5 was expressed from a single-copy plasmid under the control of the ADH1 promoter. The FLAG-tagged proteins were immunoprecipitated with anti-FLAG, and their possible co-immunoprecipitation with Cul8 and Mms1 was examined by immunoblotting with anti-HA or anti-Myc, respectively. IP, immunoprecipitation.

Interaction between Ctf4 and Mms22. A and B, co-immunoprecipitation of Ctf4 and Mms22. A, endogenous Mms22-HA was immunoprecipitated from yeast extracts, and endogenous Ctf4-FLAG that had co-immunoprecipitated was detected by immunoblotting with anti-FLAG. B, recombinant His₆HA-Mms22 and Ctf4-FLAG were expressed in Sf21 insect cells. Co-immunoprecipitation of Ctf4 with Mms22 was assessed. C, two-hybrid interactions between Mms22 and Ctf4. The L40 host strain and mms1Δ were transformed with bait (BD) plasmids and prey (AD) plasmids. Yeast strains were plated onto synthetic defined plates, with or without histidine, and with or without various amounts of 3-amino-1,2,4-triazole (3-AT) or histidine and 5-bromo-4-chloro-3-indolyl β-galactoside (X-Gal). D, identification of the Mms22- and Dia2-binding domain in Ctf4. The host strain L40 was transformed with bait (BD) plasmids and various prey (AD) plasmids containing Ctf4 deletion mutants. (The Dia2 ΔF construct is missing the F-box sequence, which increased the strength of the two-hybrid interaction (32).) The yeast strains were plated onto synthetic defined plates, with or without histidine, and with or without 15 mm of 3-AT. IP, immunoprecipitation.

Involvement of Cul8-Mms1-Mms22-Ctf4 in telomeric gene silencing. All of the strains contained URA3 at the telomeric end of chromosome VII. Yeast strains, with their genotypes indicated to the left of the figure, were grown to an A₆₀₀ of 1.0 and serially diluted in 10-fold increments, and then 4 μl of the serial dilutions was spotted onto synthetic defined plates (SD) with or without 1 μg/ml of 5-fluoroorotic acid (+5-FOA). WT, wild type.