(A) Pol2 coimmunoprecipitates with Trf4. Coexpression of FLAG-Pol2 and His-Trf4 and immunoprecipitation protocols are described in Materials and Methods. Western blots of crude extract (labeled “I” for input protein) from insect cells expressing various combinations of Trf proteins and FLAG-Pol2 were probed with antibody against Trf4 or anti-FLAG Pol2 as indicated. Lane 1, no recombinant protein; lane 2, His-Trf4; lane 3, His-Trf4 plus FLAG-Pol2. These extracts were incubated with anti-FLAG beads. After washing of the beads, proteins that bound from extracts (labeled “B” for bound protein) were eluted by boiling. The proteins were analyzed on Western blots probed with antibody against Trf4 or anti-FLAG Pol2 as indicated on the right. Lane 4, no recombinant protein; lane 5, His-Trf4; lane 6, His-Trf4 plus FLAG-Pol2. (B) Pol2 coimmunoprecipitates with Trf5. Coexpression of FLAG-Pol2 and Trf5-His is described in Materials and Methods. Western blots of crude extract from insect cells are represented in the same order as those in Fig. 2A. (C) Recombinant Trf4 prepared in E. coli stimulates Pol ɛ holoenzyme. Trf4 was purified exactly as described previously (70). The oligo(dT)_12-18 primer extension assay is described in Materials and Methods. Reaction mixtures contained 680 ng of Trf4, the amount required to observe Trf4 DNA polymerase activity (lanes 2 and 3), and/or 0.15 U of Pol ɛ, as indicated, are shown. The high level of Trf4 is saturating for stimulatory activity (see panel D). Lane 1, no protein; lane 2, Trf4-His with 0.1 mM dTTP; lane 3, Trf4-His with 1 mM dTTP; lane 4, Pol ɛ with 0.1 mM dTTP; lane 5, Pol ɛ with 1 mM dTTP; lane 6, Pol ɛ plus Trf4-His with 0.1 mM dTTP; and lane 7, Pol ɛ plus Trf4-His with 1 mM dTTP. (D) Titration of stimulatory activity of scTrf4 made in bacteria. The indicated amounts of scTrf4 were assayed for stimulation of [³H]dTMP incorporation by 0.15 U of Pol ɛ on an oligo(dT)-poly(dA) substrate as described in Materials and Methods. (E) scTrf4-His expressed in insect cell cochromatographs with Pol ɛ-stimulatory activity. scTrf4-His was expressed in insect cells. Silver staining of Trf4-His after purification through Ni²⁺-nitrilotriacetic acid and Mono Q columns, as described in Materials and Methods, and gel electrophoresis is shown at the top. Numbers refer to MonoQ fraction numbers. The same fractions are assayed for stimulation of primer extension by pol ɛ. Each fraction from the Mono Q column was dialyzed, and 2 μl of each fraction was used in a 20-μl reaction. Fraction 13 contained 17 ng of Trf4 protein (13 nM); but 2.7 nM Trf4 gave equivalent stimulation (not shown). The first lane shows no primer extension, the second lane shows activity of 0.15 U of Pol ɛ (0.5 nM) alone, and the subsequent lanes are the Mono Q fractions of the Trf4 purification assayed with 0.15 U of Pol ɛ (0.5 nm). The fraction numbers are identified above. (F) scTrf4-His and Pol ɛ-stimulatory activity copurify. [³H]dTMP incorporation assay: the same fractions from the Mono Q column were assayed for [³H]dTMP incorporation on an oligo(dT)-poly(dA) substrate as described in Materials and Methods in the presence of 0.15 U of Pol ɛ (0.5 nm). (G) Trf4 from the MonoQ column is highly purified. Coomassie-stained gel of Trf4 from fraction 14 of the MonoQ column. (H) scTrf4-His does not efficiently stimulate Pol2-140 lacking the C-terminal 1,000 amino acids. Three levels (0.075, 0.15, or 0.3 U) of either Pol ɛ (solid dots) or truncated Pol2 protein (open dots) were assayed with saturating amounts (40 ng) of Trf4-His purified from insect cells. Similar results were obtained with scTrf4 prepared in E. coli.

Effects of combining mutations in trf, pol2, and smc genes. (A) Flow cytometry profile of asynchronous cultures of exponentially growing pol2-12, trf4(896), trf5Δ, and smc1-2 single mutants. (B) Double mutants pol2-12 trf4(896) and pol2-12 trf5Δ. (C) Two pol2-12 trf4(896) trf5 triple mutants (TM6 and TM3). All strains were generated by the crosses described in Materials and Methods. Cells were grown to log phase (approximately 2 × 10⁷ cells/ml) at 30°C; however, strains exhibiting an extended S phase were less dense.

Defective sister chromatid cohesion observed in both a pol2-12 mutant and a trf5Δ mutant. (A) Flow-cytometric analysis of nocodazole-arrested pol2-12 mutant and an isogenic wild-type strain carrying the GFP signal. (B) Typical cells with attached sisters (one GFP dot per cell body) or separated sisters (two separated dots per cell body). (C) Defective sister chromatid separation was measured as total GFP dots, relative to double GFP dots (dd), for 500 to 700 cells in two separate experiments for each strain. Both pol2-12 and trf5Δ are defective. Replacing POL2 alleviates the defect. Isogenic controls for 12OG2(pADH-POL2) were wild type, SOG3, and 12OG2 (a pol2-12 mutant) (Table 1). These gave similar values of approximately 20% dd, similar to values observed with isogenic strains AFS479 and JCY122 shown here. Strains are designated as follows: AFS479 wild type (WT), JCY122 (pol2-12), JCYT59 (trf5Δ), 12OG2 [pol2-12(pADH-POL2)], JCYPD18 [cdc2-1 (Pol δ)], and JCYDNA2.

The frequency of separated sister chromatids occurring in a single nucleus increases for the pol2 mutant. Cells that displayed two GFP dots in Fig. 6 were again scored for the percentage of these that displayed two signals in one nucleus.

(A) Schematic diagram of the POL2 gene describing conserved exonuclease domains and polymerase domains. The C-terminal portion of Pol2 used for the two-hybrid library screen includes aa 1265 to 2222. Ten amino acid deletions in the zinc finger region of Pol2 were used to map the interaction of Trf5; the zinc finger deletion region is shown enlarged. (B) Schematic diagram of the TRF5 gene. The conserved nucleotidyl transferase domain occurs between aa 178 and 241. The principle conserved motif is from aa 217 to 235. Aspartates 233 and 235 in Trf5 correspond to aspartates 236 and 238 in the DXD motif in Trf4, and since mutations in these residues reduce the DNA polymerase activity of Trf4, we have labeled this the putative active site (6, 70). (C) The 78-aa POL2 interaction region of TRF5 homologous to TRF genes in yeast, human, and fly. The alignment was made using BLAST data from the SGD and GenBank. SC, S. cerevisiae; SP, Schizosaccharomyces pombe; DM, Drosophila melanogaster; HS, Homo sapiens. The corresponding amino acid numbers and accession numbers are SC-Trf5_1050861 (aa 371 to 448); SC-Trf4_950226 (aa 374 to 451); SP-C12G12.13C_2130260 (aa 1006 to 1081); DM-CG11265_22831959 (aa 461 to 538); HS-Trf4_5565687 (aa 105 to 182); HS-Lak-1_5139669 (aa 181 to 258); A. gambiae_21288943 (aa 460 to 536). Both identities and similarities are highlighted. The asterisks identify mutations in trf4 that cause lethality (trf4-378; trf4-425; trf4-444).

Genetic interaction of POL2 and TRF4. POL2 and DPB2 suppress the cold sensitivity in a trf4Δ mutant. Plasmids PDLT4, pSEY18-II, pSEY18-DPB2, and pSEY18 (GAL1 and GAL10 expression vectors with inserts of TRF4, POL2, DPB2, or empty [no insert], respectively) were transformed into strains 6265 (BY4741-trf4Δ) and the wild type (BY4741) and selected for growth on synthetic medium with dextrose-Ura. The resultant colonies were purified, grown to log phase in liquid SRaff-URA, and serially diluted onto plates containing 2% galactose. Duplicate plates were incubated at 16°C for a week.

Sensitivity of pol2-11, pol2-12, trf4, and trf5 mutants and various combination mutants to DNA synthesis inhibitors and DNA damaging reagents. Strains were grown to log phase and serially diluted at 10⁴, 10³, 10², and 10 cells per row on YPD with dimethyl sulfoxide (DMSO) (control plates, DMSO was used to dilute camptothecin; see Materials and Methods), 10-μg/ml camptothecin, 125 mM HU, and 0.03% (vol/vol) MMS. Strains are isogenic with the following designations: WT, BY4741; trf4Δ, 6265; trf5Δ, 1145; pol2-11, BY-pol2-11; pol2-12, BY-pol2-12; trf4Δ pol2-11, BY-trf4Δpol2-11; trf5Δ pol2-11, BY-trf5Δpol2-11; and trf5Δ pol2-12, BY-trf5Δpol2-12 (see Table 1).

Sister chromatid separation in a pol2 mutant begins earlier in the cell cycle than in the wild type. Wild-type cells (AFS479) and pol2-12 cells (JCY122) were grown to log phase at 30°C, induced for GFP, and then arrested with α-factor for 3 h. Cells were then released in YPD at 30°C (A and B) or 34°C (C). Samples were taken every 15 min and analyzed for bud emergence by differential interference contrast (represented as budding index), timing of sister chromatid separation by GFP assay, spindle elongation by indirect immunofluorescence using an antitubulin antibody, DNA content by flow cytometry, and nuclear morphology by DAPI staining.