Fractionation of the endogenous four subunits of DNA polymerase α in NIH 3T3 cells by glycerol density gradient centrifugation. Lysates of NIH 3T3 cells (200 μg of protein) were fractionated by 15 to 35% glycerol gradient sedimentation. The fractions were subjected to Western blotting. Protein markers run in a parallel gradient were chicken lysozyme (2.1 S), bovine serum albumin (4.4 S), yeast alcohol dehydrogenase (7.4 S), and bovine catalase (11.3 S). (A) Western blotting with antibodies specific for each subunit. (B) Densitometric analyses. Western blots were examined by scanning densitometry, and the results are presented as percentages of the maximum intensity of each signal.

Schematic representations of the results of the present study. (A) Model for the nuclear transport pathway of mouse DNA polymerase α-primase complex which shows the subcellular distribution of two, three, or four of the coexpressed subunits of DNA polymerase α. (B) Organization of the subunit assembly of DNA polymerase α. The p180 molecule can be divided into three domains. The first is the amino-terminal domain (residues 1 to 329), which is dispensable for polymerase activity and subunit assembly. The second is the core domain (residues 330 to 1279), which is sufficient for DNA polymerase activities such as template recognition, substrate binding, and the phosphoryl transfer reaction. The third is the carboxyl-terminal domain (residues 1235 to 1465), which is dispensable for polymerase activity but required for assembly of the complex. p68 binds directly to the putative zinc finger motifs in the carboxyl-terminal domain, whereas p54-p46 associates with the carboxyl-terminal domain independently of the zinc finger motifs. The interaction between p46 and p180 is mediated by p54.

Coexpression of three or four of the subunits of DNA polymerase α in COS-1 cells. (A) Western blot analysis of transfected COS-1 extracts. Two, three, or all of the subunits of DNA polymerase α were coexpressed in COS-1 cells. Forty-eight hours after transfection, the cells were lysed with a solution containing 20 mM potassium phosphate (pH 7.5), 300 mM KCl, 10% glycerol, 0.05% Triton X-100, and 0.1 mM EDTA. Ten micrograms of protein from the extract was subjected to SDS-PAGE followed by Western blot analysis with anti-p46, anti-p54, anti-p68, and anti-p180 antibodies. To express p180-p54-p46, an excess amount of pSRα180 was used because the protein level of p180 decreased considerably in the absence of p68 (23). (B) Subcellular distribution of three subunits of DNA polymerase α coexpressed in COS-1 cells. The subunits shown by green letters were detected by FITC-conjugated anti-mouse IgG antibodies. The other subunits, shown by red letters, were visualized by Texas red-conjugated anti-rabbit IgG antibody. The rightmost panels show nuclear staining by Hoechst 33258. (C) Coexpression of the four subunits. pI-pol.α was cotransfected with pI-pri or pI-pri(-NLS), which encoded the nuclear-translocation-deficient primase. Then, the subcellular distribution of ectopically expressed proteins was determined. p180 was stained by anti-p180 monoclonal antibody and FITC-conjugated anti-mouse IgG antibody. p46 was detected by anti-p46 antibody and Texas red-conjugated anti-rabbit IgG antibody. Nuclear staining with Hoechst 33258 is shown in panels c and f.

The putative zinc finger motifs in the carboxyl-terminal domain of p180 are required for the interaction with p68 but not with p54-p46. (A) Schematic representation of p180 mutant constructs. Highly conserved motifs are depicted as described in the legend to Fig. 3A. (B) Coprecipitation assay with p68 and six-His- and T7-tagged p180 mutants. Fifty micrograms of the extracts containing p68 alone (lanes 1 and 6), coexpressed p68 and six-His-tagged p180 (lanes 2 and 7), H-AZ (lanes 3 and 8), H-ZA (lanes 4 and 9), and H-AA (lanes 5 and 10) was coprecipitated with cobalt-chelating Sepharose and eluted with 200 mM imidazole. One-third of the eluates were subjected to SDS-PAGE followed by Western blot analysis with anti-p180 and anti-p68 polyclonal antibodies (lanes 6 to 10). The left panel contains 10 μg of the input proteins (lanes 1 to 5). (C) Coimmunoprecipitation assay with p54-HA-tagged p46 and six-His- and T7-tagged p180 mutants. Fifty micrograms of the extracts containing coexpressed p54-HA-tagged p46 and six-His- and T7-tagged p180 (lanes 11 and 16), H-AZ (lanes 12 and 17), H-ZA (lanes 13 and 18), H-AA (lanes 14 and 19), and H-ΔC (lanes 15 and 20) was immunoprecipitated (IP) with anti-p46 antibody. One-tenth of the precipitates were subjected to Western blot analysis with anti-T7 (upper panel of lanes 16 to 20) and anti-HA (lower panel of lanes 16 to 20) monoclonal antibodies. The left panels contain 5 μg of the input proteins (lanes 11 to 15).

Identification of the minimal core domain required for DNA polymerase activity. (A) Schematic representation of p180 mutant constructs. Highly conserved motifs are depicted as described in the legend to Fig. 3A. (B) Western blotting. COS-1 cells were transfected with constructs expressing the truncated p180 listed in panel A, and extracts were prepared at 48 h posttransfection. Five micrograms of each extract was subjected to SDS-PAGE followed by Western blot analysis with anti-T7 monoclonal antibody. Expression levels of the exogenous proteins were quantitated by densitometric scanning of the blot and are presented at the bottom as relative values compared to that of the full-length p180 (H-p180). (C) DNA polymerase activity of transfected COS-1 extracts. Five micrograms of the extracts from the transfected COS-1 cells was incubated with [³H]dTTP and DNase I-activated calf thymus DNA for 1 h at 37°C, and the incorporated radioactivity was measured. The activity of the extract from cells transfected with the vector only indicates the level of endogenous DNA polymerase activity derived from host cells. (D) DNA polymerase activities due to the exogenously expressed mutant p180 were obtained from the results shown in panel C by subtracting the endogenous DNA polymerase activity. The values were further normalized by the relative expression levels of respective proteins (obtained for panel B) and indicated as percentages of the H-p180 activity.

The cDNA expression system with IRES-derived expression plasmids. (A) Constructs of IRES-derived plasmids used for the expression of two subunits simultaneously. (B) Time course of DNA polymerase activity in the cell extracts transfected with various constructs. COS-1 cells were transfected with pcDEBΔ (■), pSRα180 alone (⧫), pSRα180 and pSRα68 (●), or pI-pol.α (▴); incubated for 24, 48, and 72 h; and then lysed with a solution containing 20 mM potassium phosphate (pH 7.5), 300 mM KCl, 10% glycerol, 0.05% Triton X-100, and 0.1 mM EDTA. After centrifugation, the supernatant was assayed to estimate DNA polymerase activity. Five micrograms of protein of COS-1 extracts was incubated with [³H]dTTP and DNase I-activated calf thymus DNA for 1 h at 37°C, and incorporation of radioactive material was measured. (C) Subcellular distribution of transiently overexpressed subunits of DNA polymerase α-primase complex. pSRα68 (a), pSRα180 (b), pSRα180 and pSRα68 (c), pI-pol.α (d), pSRα54 (e), pSRα46-HA (f), pSRα54 and pSRα46-HA (g), or pI-pri(HA) (h) was transfected into COS-1 cells, and the proteins expressed were detected by immunofluorescence analysis with anti-p68 (a, c, and d) or anti-p54 (e, g, and h) polyclonal antibodies and FITC-conjugated anti-rabbit IgG antibody or SJK132-20 anti-p180 monoclonal antibody (b, c, and d) or 12CA5 anti-HA tag antibody (f, g, and h) and Texas red-conjugated anti-mouse IgG antibody. DNA was stained blue by Hoechst 33258, and pictures were merged.

Identification of the p180 domain required for the interaction with p54-p46. (A) Schematic representation of p180 mutant constructs. H-p180, H-ΔN, H-ΔC, and H-core indicate full-length p180 with six-His tag and T7 tag at the amino terminus, amino-terminally deleted p180 with six-His tag and T7 tag at the amino terminus, carboxyl-terminally deleted p180 with six-His tag and T7 tag at the amino terminus, and both amino-terminally and carboxyl-terminally deleted p180 with six-His tag and T7 tag at the amino terminus, respectively. H-Zn, which contains only carboxyl-terminal putative zinc finger regions, is also shown for the experiments shown in Fig. 4. The seven highly conserved regions of class B DNA polymerases are indicated by blue boxes with roman numerals (I to VII) (32, 43). The five conserved regions in eukaryotic DNA polymerase α are indicated by light blue boxes with letters (A to E) (22). Putative zinc finger motifs and a putative NLS (23) are depicted by yellow boxes and a red line, respectively. Six-His and T7 tags are shown by solid boxes. Numbers indicate amino acid positions of p180. (B) Subcellular distribution of ectopically expressed p180 mutants and p54-p46. H-p180 (a to c), H-ΔN (d to f), H-ΔC (g to i), and H-core (j to l) were cotransfected with pI-pri into COS-1 cells, and the expressed proteins were detected simultaneously by indirect immunofluorescence analysis with anti-p54 polyclonal antibody and Texas red-conjugated anti-rabbit IgG antibody or anti-six-His monoclonal antibody and FITC-conjugated anti-mouse IgG antibody. Nuclear staining with Hoechst is shown in the rightmost panels. (C) Western blot analysis of transiently expressed p180 mutants. Extracts (10 μg of protein) were subjected to SDS-PAGE followed by Western blot analysis with anti-six-His monoclonal antibody. Lane numbers correspond to p180 mutant constructs shown in panel A. (D) Coimmunoprecipitation assay. Extracts (50 μg of protein) containing p54-p46 were mixed with a total of 250 μg of the extracts described for panel C (75 μg of the extracts containing H-p180, 75 μg of the extracts containing H-ΔN, 75 μg of the extracts containing H-ΔC, and 25 μg of the extracts containing H-core), incubated on ice for 2 h, and immunoprecipitated with (lane 7) or without (lane 6) anti-p46 antibody and protein G-Sepharose. One-third of the precipitates were subjected to Western blot analysis with anti-six-His monoclonal antibody. Lane 5 contains 5 μg of the input proteins. Ab, antibody; IP, immunoprecipitation.

Identification of the p180 domain required for the interaction with p68. (A) Extracts containing singly expressed p68 (50 μg of protein) were mixed in the presence (lanes 1 and 4) or absence (lanes 2 and 5) of singly expressed six-His-tagged p180 (50 μg of protein), incubated on ice for 1 h, pulled down with cobalt-chelating Sepharose (TALON), and then eluted with a solution containing 200 mM imidazole. Extracts containing coexpressed H-p180 and p68 (50 μg of protein) were also pulled down in parallel (lanes 3 and 6). One-third of the eluates were subjected to Western blot analysis with anti-p180 and anti-p68 antibodies (right panel). The left panel shows results with 5 μg of the input proteins. TL, translation. (B) Coprecipitation assay with p68 and six-His- and T7-tagged p180 mutants. Extracts containing coexpressed p68 and various mutants of the six-His- and T7-tagged p180 (50 μg of protein) were coprecipitated with cobalt-chelating Sepharose and eluted with 200 mM imidazole. One-third of the eluates were subjected to SDS-PAGE followed by Western blot analysis with anti-T7 tag monoclonal antibody (upper panels) or anti-p68 polyclonal antibody (lower panels). Precipitated protein levels of p68 were quantitated by densitometric scanning of the blot and are presented at the bottom as relative values compared to that of p68 coprecipitated with H-p180. (C) H-Zn can be expressed as a soluble form in the presence of p68. The carboxyl-terminal domain containing two zinc finger motifs was expressed alone (lanes 1 and 4) or in the presence of p54-p46 (lanes 2 and 5) or p68 (lanes 3 and 6). Transfected cells were lysed with a solution containing 20 mM potassium phosphate (pH 7.5), 300 mM KCl, 10% glycerol, 0.05% Triton X-100, and 0.1 mM EDTA, and insoluble materials were separated by centrifugation. Precipitates were resuspended in Laemmli sample buffer (17). Ten micrograms of protein of the soluble fraction and 10% of the corresponding insoluble fractions were subjected to SDS-PAGE followed by Western blotting with anti-six-His, anti-p68, and anti-p46 antibodies. (D) Coimmunoprecipitation assay with H-Zn–p68 and HA-tagged p46-p54. Fifty micrograms of the extracts containing coexpressed H-Zn–p68, coexpressed p54-HA-tagged p46, and vector control was mixed as shown on the top of the figure and immunoprecipitated (IP) with anti-p46 antibody. One-third of the precipitates were subjected to Western blot analysis with anti-T7, anti-HA, and anti-p68 antibodies. Lane 1 contains 6 μg of the extracts containing H-Zn–p68 and HA-tagged p46-p54 as input proteins.