DNA polymerase epsilon is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts

Abstract

DNA polymerase epsilon (Pol epsilon) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases alpha and delta (Pol alpha and delta), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Pol epsilon in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Pol epsilon is indeed required for chromosomal DNA replication. In Pol epsilon-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Pol alpha are loaded onto the chromatin template in Pol epsilon-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Pol epsilon, along with Pol alpha and Pol delta, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.

Figure 1

Characterization of the antibodies against the p60 subunit of Polɛ. (A) Western blotting of Xenopus egg extracts (0.5 μl) with the Polɛ p60 antibodies. (B) Coimmunoprecipitation of the catalytic subunit of Polɛ with the p60 antibodies. Immunoprecipitation from egg extracts with the p60 antibodies (αp60) (lanes 1 and 2) or with whole rabbit immunoglobulin G (control IgG) (lanes 3 and 4) was performed. The resultant supernatants (SUP, lanes 1 and 3) and precipitates (PPT, lanes 2 and 4) were analyzed by Western blotting with the p60 antibodies (Lower) or the antibodies against the catalytic subunit of Polɛ (Upper). Arrowheads indicate the catalytic subunit (p260) and p60 of Polɛ, respectively.

Figure 2

DNA replication is impaired in Polɛ-depleted extracts. (A) The Polɛ complex can be efficiently removed from egg extracts by the p60 antibodies. Five microliters of Polɛ-depleted extracts and various amounts of mock-depleted extracts were analyzed by Western blotting with the p60 antibodies. (B) Time course of DNA replication in Polɛ-depleted extracts. The amount of DNA synthesis (%) relative to that of a 100-min incubation with mock-depleted extracts is shown. (C) Replication products made with Polɛ-depleted extracts or mock-depleted extracts. The replication products of the same reactions in B were separated by neutral agarose gel electrophoresis followed by autoradiography. The position of the origin for gel electrophoresis (ori) is indicated on the right. The sizes of marker DNA are on the left. Note that the materials remaining in the gel wells are likely to be the replication intermediates because those disappeared from the wells after digestion with a single-strand DNA-specific endonuclease P1 (see Fig. 4B).

Figure 3

DNA replication can be restored by adding purified Polɛ back to the depleted extracts. (A) Purification of Xenopus Polɛ from egg extracts. An elution profile of DNA polymerase activity from Mono Q column (Top), immunoblots with Polɛ p60 or Polα p70 antibodies (Middle), and a silver-stained protein gel (Bottom) of the corresponding fractions are shown. Arrowheads indicate the positions of the catalytic subunit (p260) and p60 of Polɛ, respectively. (B) Restoration of DNA replication by the addition of purified Polɛ back to Polɛ-depleted extracts. The products from the reactions (70-min incubation) with mock- or Polɛ-depleted extracts (14 μl each) supplemented with 1 μl each of control buffer or each Mono Q fraction containing Polɛ (fraction 28 in A), Polα (fraction 23 in A), or free p60 (fraction 21 in A) were analyzed as in Fig. 2C. The position of the origin for gel electrophoresis (ori) is indicated on the right. The sizes of marker DNA are on the left.

Figure 4

Elongation of nascent DNA is retarded in Polɛ-depleted extracts. (A) Analysis of replication products by alkaline agarose gel electrophoresis. The replication reactions with Polɛ- or mock-depleted extracts were carried out for various times as indicated, and the purified products were analyzed by alkaline agarose gel electrophoresis followed by autoradiography. (B) The small DNA fragments produced in Polɛ-depleted extracts are double-stranded DNA. The replication reactions with the depleted extracts were carried out for various times as indicated. Purified products were divided in half and incubated with nuclease P1 (+) or control buffer (−). The digested products were then analyzed by neutral agarose gel electrophoresis followed by autoradiography.

Figure 5

The loading of replication proteins onto chromatin is increased in Polɛ-depleted extracts. (A and B) Western blot analysis of replication proteins bound to sperm chromatin during the incubation with Polɛ- or mock-depleted extracts. In A, sperm chromatin was incubated in the depleted extracts for 50 min in the presence of 10 μg⋅ml⁻¹ aphidicolin. In B, sperm chromatin was incubated with the depleted extracts for various times as indicated, in the presence (+Aph) or absence of aphidicolin. (C–E) The quantification of chromatin-bound Polα, RPA, and proliferating cell nuclear antigen (PCNA), respectively, in B. The amounts (%) of chromatin-bound proteins relative to those in mock-depleted extracts with aphidicolin (90-min incubation) are shown.