Dif1 Is a DNA-Damage-Regulated Facilitator of Nuclear Import for Ribonucleotide Reductase  Yang David Lee, Jun Wang, JoAnne Stubbe, Stephen J. Elledge  Molecular Cell  Volume 32, Issue 1, Pages 70-80 (October 2008) DOI: 10.1016/j.molcel.2008.08.018 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Dif1 Is an Inhibitor of S Phase Cell-Cycle Progression and a Paralog of Sml1 (A) Sequence alignment between S. cerevisiae (Sc) Dif1, Sml1, and Hug1; S. pombe (Sp) Spd1; and A. gossypii (Ag) Aer122c. The three conserved domains, the Hug domain, the Sml domain, and the Rnr1-binding domain, are boxed. (B) Simplified diagrams showing the three different domains of the orthologs of Dif1. The Rnr1-binding domain (R1B), which has been characterized through mutational analysis, was further divided into an N-terminal (cyan) and a C-terminal (blue) subdomain. (C) Synteny in the proximity of the HUG1/SML1 loci on chromosome XIII and the DIF1 (YLR437c) locus on chromosome XII in S. cerevisiae. (D) sml1Δ and mec1Δ sml1Δ strains containing vector alone or a galactose-inducible DIF1 plasmid (pGAL1::DIF1) were serially diluted and spotted on glucose and galactose media. (E) Cell-cycle profiles of sml1Δ and mec1Δ sml1Δ strains overexpressing DIF1. Cells were grown to log phase in glucose before being switched to galactose. Samples were taken from 0 to 4 hr after the galactose switch for FACS analysis of DNA content. (F) Tetrad dissection of the MEC1/mec1Δ::his5+ DIF1/dif1Δ::TRP1 diploid. Arrows mark small colonies, which are HU sensitive and tryptophan and histidine prototrophic (data not shown). Molecular Cell 2008 32, 70-80DOI: (10.1016/j.molcel.2008.08.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Dif1 Is Required for the Nuclear Localization of Rnr Small Subunits (A) dif1Δ strains complemented with either vector alone or with a plasmid containing the wild-type (WT) DIF1 gene were grown to log phase, or arrested in G1 with α factor, or arrested in G2/M with nocodazole. Cells were processed for Rnr2 visualization by indirect immunofluorescence. (B) G1- or G2/M-arrested WT and dun1Δ dif1Δ strains were processed for Rnr2 visualization by indirect immunofluorescence. (C) Comparison of Rnr2 localization in WT and wtm1Δ and dif1Δ mutants. Cells were either grown to log phase, arrested in G1, or arrested in G2/M. Samples were processed for Rnr2 visualization by indirect immunofluorescence. Examples of cells with strong nuclear Rnr2 staining are marked with white arrowheads, and cells with partially nuclear Rnr2 staining are marked with white arrows. Molecular Cell 2008 32, 70-80DOI: (10.1016/j.molcel.2008.08.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Dif1 Is Regulated during the Cell-Cycle and DNA-Damage Responses (A) Cell-cycle analysis of Dif1 abundance. Cells arrested in G1 by α factor were released into the cell cycle. Samples were collected at 10 min intervals for western blotting of Dif1. Clb5 was used to monitor cell-cycle progression. Ponceau staining served as a loading control. A log phase sample from an asynchronous (AS) population of cells was loaded in the first lane for comparison. (B) Western blot analysis of Dif1 from log phase wild-type (WT) and dun1Δ cells treated with HU (150 mM, 1.5 hr) or MMS (0.1%, 1.5 hr). Tubulin (Tub1) serves as a loading control. (C) Western blot analysis of Dif1 from WT and dun1Δ cells treated with phleomycin (Ph, 50 ng/ml, 1 hr) or ionizing radiation (IR, 20 kRad, 1 hr) while maintained in G2/M arrest. (D) Mec1-dependent posttranslational modification of 3Myc-Dif1 in sml1Δ and mec1Δ sml1Δ cells. Cells with a single copy of 3Myc-DIF1 integrated at the endogenous locus were arrested in G1 by α factor, and then released into the cell cycle in the presence of HU (150 mM). Samples were collected at 20 min intervals for western analysis of 3Myc-Dif1 mobility and abundance. Clb5 was used to monitor cell-cycle progression. (E) Phosphatase (PPase) treatment of 3Myc-Dif1. Log phase yeast cells carrying integrated 3Myc-DIF1 were treated with HU (150 mM, 1.5 hr). 3Myc-Dif1 was immunoprecipitated from the lysate and subjected to PPase treatment, with or without PPase inhibitors. (F) Dun1-dependent Dif1 phosphorylation after HU treatment. WT and dun1Δ strains with integrated 3Myc-DIF1 were arrested in G1 by α factor, and then released into HU media (150 mM). Samples were collected at 20 min intervals for western analysis of 3Myc-Dif1 mobility and abundance. (G) Rnr2 visualization by indirect immunofluorescence of WT or a 3Myc-DIF1-integrated strain arrested in G1 phase with α factor, and then released into HU media (150 mM) for 1 and 2 hr. (H) Phosphomapping of 3Myc-Dif1. Log phase cells with integrated 3Myc-DIF1 were treated with HU (150 mM, 1.5 hr). 3Myc-Dif1 was immunoprecipitated with anti-Myc antibodies, resolved and silver stained on SDS-PAGE, and excised for phosphomapping by mass spectrometry. The “#” signs indicate two potential sites for phosphorylation. Molecular Cell 2008 32, 70-80DOI: (10.1016/j.molcel.2008.08.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 The Sml and Hug Domains Have Distinct Roles in Dif1 Regulation (A) Western blot analysis of wild-type (WT) and phospho mutant Dif1 after phleomycin treatment. dif1Δ strains containing a WT DIF1 plasmid (pDIF1-WT) or the phospho mutant plasmid (pDIF1-4A) were treated with phleomycin (50 ng/ml) for the indicated times during G2/M arrest by nocodazole. Tubulin (Tub1) served as a loading control. (B) Western blot analysis of WT and phospho mutant Dif1 from cells arrested in G1 by α factor, and then released into HU media (150 mM) for the indicated times. (C) G2/M-arrested WT and phospho mutant Dif1 strains were treated with phleomycin for 2 hr and processed for Rnr2 visualization by indirect immunofluorescence. (D) WT or phospho mutant Dif1 cells were arrested in G1 by α factor (αF) or released from G1 into HU (150 mM) for 1 hr and processed for Rnr2 visualization by indirect immunofluorescence. Examples of cells with Rnr2 staining that is predominately nuclear (white arrowhead), predominately cytoplasmic (black arrow), or equal in distribution (white arrow) are indicted. (E) Quantification of (D). For each treatment, more than 160 cells were counted (range: 161–200 cells). (F) Western blot analysis of WT, hug-domain mutant, or sml-domain mutant Dif1 after phleomycin treatment. dif1Δ strains complemented by plasmids containing WT (pDIF1-WT), hug-domain mutant (pDIF1-hug), or sml-domain mutant (pDIF1-sml) versions of DIF1 were treated with phleomycin (50 ng/ml) for the indicated times while maintained in G2/M. Tubulin (Tub1) served as the loading control. (G) Western blot analysis of Dif1, Dif1-hug, and Dif1-sml mutants after HU treatment. dif1Δ strains complemented by plasmids containing WT (pDIF1-WT), hug-domain mutant (pDIF1-hug), or sml-domain mutant (pDIF1-sml) versions of DIF1 were arrested in G1 by α factor, and then released into HU media (150 mM) for the indicated times. (H) Rnr2 visualization by indirect immunofluorescence from log phase cultures of dif1Δ strains carrying pDIF1-WT or pDIF1-sml plasmids. (I) Dun1 in vitro kinase assay. Polyclonal anti-Dif1 antibodies were used to immunoprecipitate Dif1 from dif1Δ yeast strain complemented by plasmid alone (Δ), wild-type (WT), phospho mutant (4A), and hug-domain mutant (hug) Dif1, and then incubated with or without recombinant GST-Dun1 purified from insect cells in the presence of [γ32-P]ATP. The bottom panel shows a western blot with anti-Dif1 antibodies. The predominant Dif1 bands are marked with single asterisks. The difference in their size is due to site-directed mutagenesis. The top panel shows an autoradiograph; the expected position of each Dif1 from the western blot is marked by arrows. The nonspecific bands present even in the dif1Δ sample are marked by double asterisks. Molecular Cell 2008 32, 70-80DOI: (10.1016/j.molcel.2008.08.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Dif1 Is Much Less Abundant than the Rnr2-Rnr4 Heterodimer (A) Left panel: known standards of bovine serum albumin (BSA) were resolved on SDS-PAGE alongside of five-fold serial dilutions of purified recombinant His6-Dif1 to estimate the concentration of the original Dif1 sample in nanograms. Dif1 serial dilutions that are beyond the range of the BSA standard were not used for estimation of protein concentration and were marked by asterisks (∗). Right panel: known quantities of His6-Dif1 were resolved on SDS-PAGE alongside of lysates from asynchronous (AS) and synchronized WT cells released from a G1 block and taken at 20 min intervals. The membrane was probed with anti-Dif1 antibodies. (B) As in (A), except measuring Rnr1. Right panel: estimated Rnr1:Dif1 ratios are indicated below the western blot for the asynchronous, 0 min, and 80 min time points. (C) As in (A), except measuring Rnr2. (D) As in (A), except measuring Rnr4. (E) Clb5 western blot to show progression of S phase in the synchronized culture. (F) Tubulin western blot serves as a loading control. Molecular Cell 2008 32, 70-80DOI: (10.1016/j.molcel.2008.08.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Dif1 Controls the Nuclear Import of Rnr2-Rnr4 (A) The nuclear localization of Rnr2 in the dif1Δ wtm1Δ double mutant was compared to wild-type (WT), wtm1Δ, and dif1Δ single mutants. Log phase and G1-arrested cells were processed for visualization of Rnr2 by indirect immunofluorescence. Cells with strong nuclear Rnr2 staining (white arrowhead), residual nuclear Rnr2 staining (white arrow), and Rnr2 nuclear exclusion staining (black arrow) are indicated. (B) wtm1Δ strains containing vector alone or a galactose-inducible DIF1 plasmid (pGAL::DIF1) were grown to log phase in glucose media. Cells were then switched to galactose media for 3 hr, while either being kept in log phase or arrested in G1 or G2/M. Samples were processed for Rnr2 visualization by indirect immunofluorescence. (C) A schematic of the experiment used to examine nuclear import of Rnr2-Rnr4. Cells were grown to log phase in raffinose-glucose media, then switched to raffinose-galactose media for 3 hr to turn on the transcription of the GAL1-GFP-RNR4 reporter. The promoter was shut off by switching back to glucose media for 3 hr, before leptomycin B was added for an additional hour (LMB, 200 ng/ml). For the analysis of G1-arrested cells, the last two steps (4 hr) were carried out in the presence of α factor. Cells were photographed for GFP-Rnr4 localization. (D) GFP-Rnr4 localization in WT, wtm1Δ, dif1Δ, and wtm1Δ dif1Δ mutants in the LMB-sensitive (Crm1-T539C) background were treated with or without LMB during log phase. (E) Quantification of GFP-Rnr4 localization in (D). (F) Quantification of GFP-Rnr4 localization in G1-arrested cells, treated with or without LMB. Molecular Cell 2008 32, 70-80DOI: (10.1016/j.molcel.2008.08.018) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Dif1 Directly Interacts with Rnr2-Rnr4 (A) Binding between Dif1 and Rnr2-Rnr4 was detected with BIAcore. Purified Rnr2-Rnr4 was immobilized on the sensor surface. Recombinant wild-type (WT) Dif1 protein (Dif1-WT) flowed over the immobilized Rnr2-Rnr4 at the indicated concentrations. Recombinant Dif1-hug (Dif1-hug) protein flowed over the same sensor surface at a concentration of 5.0 μM (turquoise line). Binding between Rnr2-Rnr4 and Dif1 was measured in response units (RU). (B) Diagram of the amino acid changes in the Dif1-hug mutant. (C) Rnr2 visualization by indirect immunofluorescence from log phase cultures of dif1Δ strains carrying pDIF1-WT or pDIF1-hug plasmids. (D) Model demonstrating nuclear import and retention of Rnr2-Rnr4 in response to DNA damage. (Left) Dif1 facilitates the nuclear import of Rnr2-Rnr4, whereas Wtm1 functions as a nuclear anchor for Rnr2-Rnr4. In the absence of DNA damage, the net contribution of these two pathways leads to a net accumulation of Rnr2-Rnr4 inside the nucleus. (Right) Activation of the DNA-damage response (DDR) leads to the phosphorylation, inactivation, and degradation of Dif1, reducing the nuclear import of Rnr2-Rnr4. DNA damage also releases the pool of nuclear Rnr2-Rnr4, either by decreasing the affinity between the Wtm1-Rnr2-Rnr4 interactions, or through an increased rate of Crm1-mediated Rnr2-Rnr4 nuclear export. Molecular Cell 2008 32, 70-80DOI: (10.1016/j.molcel.2008.08.018) Copyright © 2008 Elsevier Inc. Terms and Conditions