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Published in final edited form as: DNA Repair (Amst). 2008 Jan 31;7(3):439–451. doi: 10.1016/j.dnarep.2007.11.016

(A) In the yeast two-hybrid assay, S. cerevisiae Rev1 interacts with Rev7, but not Rad30 (Polη). (B) In S. cerevisiae, tagged Rad30 (Polη) is fully functional for UV radiation survival: comparison of WT (W1588-4C), Rad30-TEV-ProA-His (RWY13), and rad30Δ (RWY15) strains. Error bars represent standard error. (C) Rev1-HA-pAS311 can rescue the MMS sensitivity of the rev1 null mutant. Top row: wildtype (W1588-4A + pAS311), second row: rev1Δ (YLW20 + pAS311) bottom row: Rev1-HA (YLW20 + pAS311-REV1-HAC (D) S. cerevisiae Rev1-HA coimmunoprecipitates endogenously tagged Rev7-Myc. Lane 1: Rev1-Cterm-HA and Rev7-13Myc (YSD5 + pAS311-REV1CT239-HAC); Lane 2: Full length Rev1-HA and Rev7-13Myc (YSD5 + pAS311-Rev1-HAC); Lane 3: Rev7-13Myc alone (YSD5 + pAS311). Full length Rev1 is produced at lower levels than the C-terminal 239 amino acid fragment, resulting in the difference in quantity of Rev7-Myc which coIPs in lane 1 compared with lane 2. (E) In S. cerevisiae, endogenously tagged Rev7 immunoprecipitates Rev1-HA. Rev7-13Myc immunoprecipitates Rev1 in the presence (+) of Rev1-HA (YSD5 + pAS311-Rev1-HAC) but does not in the absence (−) of Rev1-HA (YSD5 + pAS311). Rev1-HA is undetectable in the input (not shown). (F) S. cerevisiae Rev1 and Rad30 (Polη) do not co-immunoprecipitate in the presence or absence of UV-damage. IgG was used to precipitate Rad30-TEV-ProA-7His protein using the alternate coIP protocol with strains RWY75 and YSD7. Lane 1: RWY75 input sample probed with PAP for Rad30-TEV-ProA-7His; Lane 2: RWY75 input; Lane 3, YSD7 IP; Lane4, RWY75 IP, showing Rad30 band only. Lanes 2–4 probed with anti-HA antibody, detects Rev1-HA present in the input and also (through the IgG-binding activity of ProA) nonspecifically detects the high concentration of Rad30-ProA in the IP. For UV-treated conditions, yeast extracts were made from cells that had been subjected to UV radiation. IgG was used to precipitate Rad30-TEV-ProA-7His protein by the primary coIP protocol. Lane 1: Rev1-HA and Rad30-ProA (RWYRWY254 + pAS311-REV1-HAC); Lane 2: Rev1-HA only (RWY270 + pAS311-REV1-HAC); Lane 3: Rad30-ProA only (RWY254).

(A) In the yeast two-hybrid assay, S. cerevisiae Rev1 interacts with Rev7, but not Rad30 (Polη). (B) In S. cerevisiae, tagged Rad30 (Polη) is fully functional for UV radiation survival: comparison of WT (W1588-4C), Rad30-TEV-ProA-His (RWY13), and rad30Δ (RWY15) strains. Error bars represent standard error. (C) Rev1-HA-pAS311 can rescue the MMS sensitivity of the rev1 null mutant. Top row: wildtype (W1588-4A + pAS311), second row: rev1Δ (YLW20 + pAS311) bottom row: Rev1-HA (YLW20 + pAS311-REV1-HAC (D) S. cerevisiae Rev1-HA coimmunoprecipitates endogenously tagged Rev7-Myc. Lane 1: Rev1-Cterm-HA and Rev7-13Myc (YSD5 + pAS311-REV1CT239-HAC); Lane 2: Full length Rev1-HA and Rev7-13Myc (YSD5 + pAS311-Rev1-HAC); Lane 3: Rev7-13Myc alone (YSD5 + pAS311). Full length Rev1 is produced at lower levels than the C-terminal 239 amino acid fragment, resulting in the difference in quantity of Rev7-Myc which coIPs in lane 1 compared with lane 2. (E) In S. cerevisiae, endogenously tagged Rev7 immunoprecipitates Rev1-HA. Rev7-13Myc immunoprecipitates Rev1 in the presence (+) of Rev1-HA (YSD5 + pAS311-Rev1-HAC) but does not in the absence (−) of Rev1-HA (YSD5 + pAS311). Rev1-HA is undetectable in the input (not shown). (F) S. cerevisiae Rev1 and Rad30 (Polη) do not co-immunoprecipitate in the presence or absence of UV-damage. IgG was used to precipitate Rad30-TEV-ProA-7His protein using the alternate coIP protocol with strains RWY75 and YSD7. Lane 1: RWY75 input sample probed with PAP for Rad30-TEV-ProA-7His; Lane 2: RWY75 input; Lane 3, YSD7 IP; Lane4, RWY75 IP, showing Rad30 band only. Lanes 2–4 probed with anti-HA antibody, detects Rev1-HA present in the input and also (through the IgG-binding activity of ProA) nonspecifically detects the high concentration of Rad30-ProA in the IP. For UV-treated conditions, yeast extracts were made from cells that had been subjected to UV radiation. IgG was used to precipitate Rad30-TEV-ProA-7His protein by the primary coIP protocol. Lane 1: Rev1-HA and Rad30-ProA (RWYRWY254 + pAS311-REV1-HAC); Lane 2: Rev1-HA only (RWY270 + pAS311-REV1-HAC); Lane 3: Rad30-ProA only (RWY254).