The DNA polymerase activity of Pol epsilon holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts

Abstract

Background: DNA polymerase epsilon (Pol epsilon) is involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the roles of replicative Pol alpha and Pol delta in chromosomal DNA replication are relatively well understood and well documented, the precise role of Pol epsilon in chromosomal DNA replication is not well understood.

Results: This study uses a Xenopus egg extract DNA replication system to further elucidate the replicative role(s) played by Pol epsilon. Previous studies show that the initiation timing and elongation of chromosomal DNA replication are markedly impaired in Pol epsilon-depleted Xenopus egg extracts, with reduced accumulation of replicative intermediates and products. This study shows that normal replication is restored by addition of Pol epsilon holoenzyme to Pol epsilon-depleted extracts, but not by addition of polymerase-deficient forms of Pol epsilon, including polymerase point or deletion mutants or incomplete enzyme complexes. Evidence is also provided that Pol epsilon holoenzyme interacts directly with GINS, Cdc45p and Cut5p, each of which plays an important role in initiation of chromosomal DNA replication in eukaryotic cells.

Conclusion: These results indicate that the DNA polymerase activity of Pol epsilon holoenzyme plays an essential role in normal chromosomal DNA replication in Xenopus egg extracts. These are the first biochemical data to show the DNA polymerase activity of Pol epsilon holoenzyme is essential for chromosomal DNA replication in higher eukaryotes, unlike in yeasts.

Figure 1

p12, p17 and p60 of Xenopus Pol ε interact with the C-terminal region of p260. (A) Schematic representation of wild type (full)- and mutant (ΔC2157, ΔC1054, Δ860N, and ΔCat) forms of xPol ε p260. The conserved catalytic DNA polymerase domain and putative zinc finger domain of Pol ε are indicated. (B) FLAG-tagged p260 was co-expressed with p60, p17 and p12 in insect cells. Cell lysates were prepared and immunoprecipitated with anti-FLAG antibody and the precipitates were subjected to SDS-PAGE followed by immunoblotting with antibodies for each subunit. "Lysates" and "bound" indicate total protein and immunoprecipitated proteins, respectively. (C) Schematic representation of Xenopus Pol ε holoenzyme.

Figure 3

Interactions between histidine-tagged p260 (H-p260) or p60 (H-p60) and other subunits of Pol ε. Interactions between histidine-tagged p260 (H-p260) or p60 (H-p60) and other subunits of Pol ε were investigated using a pull down assay with Ni²⁺ -chelating beads and crude extracts prepared from insect cells expressing the indicated subunits of xPol ε. Cell lysates containing H-p260 or H-p60 (lane 1) and bound proteins (lane 2) were subjected to SDS-PAGE, followed by immunoblotting with the indicated antibodies. Beads without Ni²⁺ were also used as a control (lane 3).

Figure 2

Purified xPol ε holoenzyme from insect cells. Wild type and mutant FLAG-tagged p260 was co-expressed with p60, p17 and p12 in insect cells and Pol ε complexes were purified by DEAE Sepharose and anti-FLAG antibody chromatography. Fractions were eluted from the antibody affinity column, pooled and subjected to SDS-PAGE followed by CBB staining (Left) and immunoblotting (Right). Native Xenopus Pol ε (native) holoenzyme was purified from Xenopus egg extracts as described previously [22]. Fractions shown are rPol ε, rDN Pol ε, rΔcat Pol ε, and rF260 (see text). rF260 is FLAG-tagged p260.

Figure 4

DNA replication activity in Pol ε-depleted Xenopus egg extracts. (A) The same number of wild type-, mutant or partial r-xPol ε complex molecule, which has been estimated based on the result of Fig. 2, was added to xPol ε-depleted egg extracts as indicated (see text for detailed description of mutants and partial complexes). DNA replication was initiated by the addition of sperm chromatin and the rate and extent of DNA synthesis was measured by neutral agarose gel electrophoresis followed by autoradiography [22]. The origin (well) is and DNA size markers are indicated on the right. (B) Radioactive material, which migrated slower than the 23-kb marker DNA (shown by an arrow in (A)), was considered to be fully replicated product and quantified by a scintillation counter.

Figure 5

Xenopus Pol ε holoenzyme interacts directly with xGINS, xCdc45p, and xCut5p in vitro. (A) Flag-tagged r-xPol ε holoenzyme was incubated with xGINS [29] for 1 h at 4°C and immunoprecipitated with anti-Flag antibody. The immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting. IP; immunoprecipitates, SUP; supernatant. (B) Increasing concentrations of xCut5p were incubated with xGINS and Flag-tagged xCdc45 [30] or Flag-tagged BAP for 1 h at 4°C and immunoprecipitated as in (A). (C) r-xPol ε holoenzyme was pre-incubated with either Flag-tagged xGINS or Flag-tagged xCdc45p prior to addition of the indicated protein(s). Reactions were analyzed as in (A). (D) A model for the interaction between r-xPol ε and replication accessory proteins (xGINS, xCdc45, and xCut5).

Figure 6

xGINS stimulates DNA synthesis by xPol ε holoenzyme. (A) DNA synthesis reactions (10 μl) contained 200 fmol ³²P-labeled 34-mer primer/65-mer template replication substrate [32], 15 fmol r-xPol ε holoenzyme and 45 fmol (×5), 150 fmol (×10), or 300 fmol (×20) xGINS as indicated. Reactions were incubated at 25°C for the indicated amount of time, terminated by addition of stop solution (5 μl), and analyzed by sequencing gel and autoradiography [32]. The ³²P-labeled 65-mer is the reaction product and the ³²P-labeled 34-mer is the primer.(B) The amount of the reaction products in (A) (65-mer) was quantified by Image analyzer (Fiji).