The fifth essential DNA polymerase phi in Saccharomyces cerevisiae is localized to the nucleolus and plays an important role in synthesis of rRNA

Abstract

We report that POL5 encodes the fifth essential DNA polymerase in Saccharomyces cerevisiae. Pol5p was identified and purified from yeast cell extracts and is an aphidicolin-sensitive DNA polymerase that is stimulated by yeast proliferating cell nuclear antigen (PCNA). Thus, we named Pol5p DNA polymerase phi. Temperature-sensitive pol5-1-- -3 mutants did not arrest at G(2)/M at the restrictive temperature. Furthermore, the polymerase active-site mutant POL5dn gene complements the lethality of Delta pol5. These results suggest that the polymerase activity of Pol5p is not required for the in vivo function of Pol5p. rRNA synthesis was severely inhibited at the restrictive temperature in the temperature-sensitive pol5-3 mutant cells, suggesting that an essential function of Pol5p is rRNA synthesis. Pol5p is localized exclusively to the nucleolus and binds near or at the enhancer region of rRNA-encoding DNA repeating units.

Figure 1

Pol5p has Pol activity. Yeast YKS1 cells harboring pYEUra3-POL5 were grown in YGP, lysed, and analyzed by SDS/PAGE. (A) After SDS/PAGE, the gel was stained with Coomassie blue. Lane 1, control vector, +galactose; lane 2, control vector, +glucose; lane 3, pYEUra3-POL5 DNA expression plasmid, +galactose; lane 4, pYEUra3-POL5 DNA, +glucose; lane 5, pYEUra3-POL5dn, +galactose; lane 6, pYEUra3-POL5dn, +glucose. (B) After SDS/PAGE, an in situ gel assay for Pol activity was carried out as published (9, 11). Lane 1, 10 units of T4 Pol; lane 2, pYEUra3-POL5 expression plasmid, +glucose; lane 3, pYEUra3-POL5 expression plasmid, +galactose; lane 4, pYEUra3-POL5dn, +galactose. (C) Recombinant GST-Pol5p was purified from DY150 containing pYEX4T-POL5 cells induced with CuSO₄ and analyzed by SDS/PAGE and Western blotting. Lane 1, Coomassie blue staining; lane 2, Western blot with mouse antiserum against GST-Pol5p. (D) Purification of Pol5p from yeast cells. The figure shows the Pol activity profile from Mono S column. The Pol active fractions were subjected to SDS/PAGE followed by Western blotting with mouse antiserum against GST-Pol5p or rabbit antiserum against the Polɛ complex. (E) Pol active fractions from the Mono Q column (lanes 20–22) were analyzed by SDS/PAGE and Coomassie blue staining.

Figure 2

Putative PCNA-binding consensus sequences in Pol5p. The region between residues 328 and 343 of S. cerevisiae Pol5p (ScPol5p) has high homology to one of the PCNA-binding domains in ScPol32p. The figure shows the amino acid sequences of ScPol5p, SpPol5p, ScPol32p, and ScCdc7p (Pol5 consensus). The red-colored shaded amino acids are identical amino acid residues.

Figure 3

(A) S. cerevisiae Pol5p has the six Pol domains that are conserved among B-type Pols. It also has the acidic region (orange color) and many leucine-charged domains (light green and dark green) found in human MybBP1A. Shown is one of six Pol domains (domain I) that are most conserved among B-type Pols (a red square) and the mutation sites found in pol5-1∼–3 mutants in Pol5p. (B) Amino acid sequence similarity between ScPol5p, SpPol5p, and human MybBP1A (Hs MybBP1A) and between ScReb1p, SpReb1p, and human c-Myb (Hs c-Myb). It is known that Hs MybBP1A physically interacts with Hs c-Myb.

Figure 4

Pol5p is required for rRNA synthesis but not for chromosomal DNA replication. YSK13 (pol5-3) cells were grown in SD-complete without tryptophan at 25°C to ≈1 × 10⁶ cells per ml. The culture was divided into two portions and incubated at 25 and 37°C, respectively. Aliquots were analyzed for cell growth (A) or FACScan analysis (B). (C) YSK14 harboring YEplac195-POL5 was transformed by YCplac22-POL5dn and grown on either YPD- or SD-complete tryptophan plate with 5-fluoroorotic acid at 25°C for 4 days. When YEplac195-POL5 plasmid was removed from the strain (+5-fluoroorotic acid), YSK14 (Δpol5) harboring YCplac22-POL5dn was able to grow. (D) YSK10 (POL5) and YSK13 (pol5-3) cells were grown in 50 ml of SD complete without tryptophan to ≈1 × 10⁶ cells per ml at 25°C. [³H]uridine [250 μCi (1 Ci = 37 GBq)] was added to the culture, and incubation continued for 60 min. Then the culture was divided into two equal portions, which were incubated at 25 or 37°C. Aliquots were withdrawn for analysis of ³H-labeled RNA and DNA (24). (E) YSK10 (POL5) and YSK13 (pol5-3) mutant cells were grown in 50 ml of SD complete without tryptophan to ≈5 × 10⁶ cells per ml at 25°C and divided into two equal portions, which were incubated at 25 or 37°C. For a control, cells were pulse-labeled for 15 min before temperature-shift up. At 30, 60, and 120 min after temperature shift, cells were pulse-labeled with 30 μCi/ml of [³H]uridine for 15 min, and total RNA was extracted (35) and analyzed by 2.5% agarose gel electrophoresis. After electrophoresis, the gel was stained with ethidium bromide and photographed (EtBr Staining). The labeled RNA was transferred onto nitrocellulose and autoradiographed by using a Fuji imaging plate for 8 days (Autoradiography). Lanes 1, 2, 3, and 4 are RNA from either wild-type (POL5) or pol5-3 mutant cells pulse-labeled with [³H]uridine for 15 min at 0, 30, 60, and 120 min, respectively, after being grown at 25°C. Lanes 5, 6, 7, and 8 are RNA from either POL5 or pol5-3 mutant cells pulse-labeled with [³H]uridine for 15 min at 0, 30, 60, and 120 min, respectively, after temperature shift to 37°C.

Figure 5

Pol5p is localized exclusively in the nucleolus. (A) Yeast W303-1A cells expressing 9Myc-tagged Pol5p were grown in 10 ml of YPD, harvested by centrifugation, and fixed with formaldehyde, and Pol5p and Nop1p were stained with monoclonal antibody to Myc or Nop1p. Ph, photograph of cells; DNA, 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei; Pol5p and Nop1p, fluorescence images from immunoreaction with Myc and Nop1p antibodies, respectively. Nop1p + DNA, Pol5p + DNA, Pol5p + Nop1p, and Pol5p + Nop1p + DNA are the merged images of Pol5p and DNA, Pol5p and Nop1p, Nop1p and DNA, and Pol5p, Nop1p, and DNA, respectively. An arrow indicates ×5-enlarged image. (B) YSK13 (pol5-3) cells expressing 9Myc-tagged pol5-3 were grown at 25°C and divided to two equal portions, and each was incubated at 25 or 37°C for another 2 h. Then, cells were fixed and stained with DAPI and Myc or Nop1p antibody as described for A. Photograph of cells (Ph) or the merged image of DAPI (blue) and Myc or Nop1p monoclonal antibody staining (red) is shown. (C) Pol5p does not interact physically with Nop1p in the nucleolus. Yeast W303-1A cells expressing 9Myc-tagged Pol5p were grown in 100 ml of YPD, harvested by centrifugation, and resuspended in 5 ml of the lysis buffer. Protein extracts were prepared and subjected to immunoprecipitation by using anti-Myc monoclonal antibody as described (27). The immunoprecipitates were analyzed by SDS/PAGE followed by immunoblotting with anti-Myc and anti-Nop1p monoclonal antibodies. As a control, no Myc-tagged W303-1A cell extracts were used. (a) Protein extracts prepared from W303-1A cells expressing 9Myc-tagged Pol5p without treatment of the dithiobis (succinimidylpropionate) (DSP) cross-linker. (b) Protein extracts from W303-1A cells expressing 9Myc-tagged Pol5p with treatment of DSP prior to protein extraction. E and IP, whole extracts and immunoprecipitates by anti-Myc monoclonal antibody, respectively; + and −, protein extracts from cells expressing 9Myc-tagged Pol5p and those from non-tagged cells, respectively. (D) Pol5p may bind on/near the enhancer sequence located in the rDNA repeating unit. Yeast W303-1A cells expressing 9Myc-tagged Pol5p were grown in 100 ml of YPD medium, fixed with formaldehyde, harvested by centrifugation, and resuspended in 5 ml of the lysis buffer. Protein extracts were prepared, sonicated, and subjected to immunoprecipitation by using anti-Myc polyclonal antibodies. DNA was extracted from the immunoprecipitates and amplified by PCR using specific primer sets for the enhancer region (E, 294 bp), autonomously replicating sequence region (ARS, 331 bp), and ≈4 kb away from the enhancer sequence (E + 4 kb, 269 bp). The DNA fragments amplified were analyzed by agarose gel electrophoresis. WCE, DNA from whole-cell extracts; + and −, with and without Myc tag or formaldehyde cross-linking, respectively. (D Upper) Schematic representation of the structure of the rDNA repeating unit of yeast chromosomal DNA.

Figure 6

The size of chromosome XII increases in pol5 mutants. Wild-type (wt, W303-1A), temperature-sensitive pol5-1∼–3 mutants, and the Δpol5 strain containing the POL5dn gene (pol5dn) were grown at 25°C, and their chromosomal DNA was extracted and subjected to pulse-field agarose gel electrophoresis as published (14, 28). DNA was strained with ethidium bromide and photographed. Roman numbers indicate the chromosome number of S. cerevisiae.