Deletion of IXR1 leads to increased Rnr3 and Rnr4 levels and decreased Sml1 levels.

(A) Western blot analysis of Rnr3-HA and Rnr4 levels in wild-type (AC447-2A), ixr1-S366F (TOY619), and ixr1Δ (TOY621) strains before and after 2 hours treatment with 0.2 mg/L 4-nitroquinoline 1-oxide (4-NQO), 200 mM HU, or 0.02% methyl methanesulfonate (MMS). Rnr3 and Rnr4 levels were quantified in relative units (RU, levels of Rnr3 or Rnr4 divided by the levels of tubulin in corresponding sample) as described in Materials and Methods. ND – not detected. (B) Western blot analysis of Rnr2 levels in wild-type (AC447-2A) and ixr1Δ (TOY621) strains before treatment and after 2 hours treatment with 0.2 mg/L 4-NQO, 0.02% MMS, or 200 mM HU. (C) Western blot analysis of Sml1 levels in wild-type (W1588-4C) and ixr1Δ (TOY736) strains before treatment and after 2 hours treatment with 0.02% MMS. (D) Western blot analysis of Rad53 phosphorylation status in wild-type (W1588-4C) and ixr1Δ (TOY736) strains before treatment and after 2 hours treatment with 0.02% MMS. (E) Deletion of RAD53 but not of SML1 or DUN1 abolishes the upregulation of Rnr3 and Rnr4 levels in ixr1Δ. Western blot analysis of Rnr3-HA and Rnr4 levels. The following strains were analyzed: wt (AC447-2A), ixr1Δ (TOY732), ixr1Δ sml1Δ (TOY778), ixr1Δ dun1Δ sml1Δ (TOY772), and ixr1Δ rad53Δ sml1Δ (TOY781).