Schizosacchromyces pombe Dpb2 binds to origin DNA early in S phase and is required for chromosomal DNA replication

Abstract

Genetic evidence suggests that DNA polymerase epsilon (Pol epsilon) has a noncatalytic essential role during the early stages of DNA replication initiation. Herein, we report the cloning and characterization of the second largest subunit of Pol epsilon in fission yeast, called Dpb2. We demonstrate that Dpb2 is essential for cell viability and that a temperature-sensitive mutant of dpb2 arrests with a 1C DNA content, suggesting that Dpb2 is required for initiation of DNA replication. Using a chromatin immunoprecipitation assay, we show that Dpb2, binds preferentially to origin DNA at the beginning of S phase. We also show that the C terminus of Pol epsilon associates with origin DNA at the same time as Dpb2. We conclude that Dpb2 is an essential protein required for an early step in DNA replication. We propose that the primary function of Dpb2 is to facilitate assembly of the replicative complex at the start of S phase. These conclusions are based on the novel cell cycle arrest phenotype of the dpb2 mutant, on the previously uncharacterized binding of Dpb2 to replication origins, and on the observation that the essential function of Pol epsilon is not dependent on its DNA synthesis activity.

Figure 1.

Protein sequence alignment of S. pombe dpb2⁺, S. cerevisiae DPB2, and human DPE2. Fission yeast dpb2⁺ shares 31% identity with both the human and S. cerevisiae genes. Identical residues are indicated in black, whereas homologous residues are indicated in gray. Sequence alignment was performed using ClustalW 1.8.

Figure 2.

dpb2⁺ is essential for cell viability. (A) Strategy for deleting dpb2⁺ by gene replacement. The 1.76-kb open reading frame of dpb2⁺ was replaced with the 1.8-kb ura4⁺ gene. A HindIII fragment containing the ura4⁺ gene was cloned into pBluescript. A 1.2- and 2-kb fragment corresponding to sequences upstream and downstream of the dpb2⁺ open reading frame were cloned into the EcoRI/HindIII and HindIII/XhoI sites of pBluescript-ura4⁺, respectively. The resulting plasmid was digested with EcoRI and XhoI and the excised fragment was transformed into the diploid strain GD28. Positive transformants were selected on minimal medium minus uracil. (B) Southern blot analysis of heterozygous diploid +/Δ (lanes 1 and 2) and homozygous diploid +/+. DNA was digested by NdeI and Bsu36I and probed with the EcoRI and HindIII fragment. (C) Tetrad analysis of the heterozygous diploid strain (+/Δ) revealed a two viable:two nonviable segregation as expected for an essential gene disruption. All viable spores were confirmed to be ura^–.

Figure 3.

Cells depleted for Dpb2 are defective in S phase progression. (A) A culture of haploid GD149 cells was grown to log phase, the culture was then diluted and one-half was treated with 10 μg/μl thiamine to repress dpb2⁺ gene expression. Samples were collected at the indicated times and DNA content analyzed by flow cytometry. (B) Cells stained with the DNA binding dye 4,6-diamidino-2-phenylindole after incubation with thiamine at 0, 23, and 40 h. Arrows indicate abnormal nuclear morphology, including missegregated chromosomes and anucleate cells. (C) DNA replication initiation is delayed in cell cycle synchronized cultures depleted for Dpb2. Wild-type (972) or GD149 cells were grown in the absence of nitrogen to arrest cells in G1. During nitrogen starvation, thiamine was added (10 μg/μl) to the media to repress transcription from the nmt81 promoter. After 20 h or when >90% of the cells showed a G1 arrest, cells were induced to reenter the cell cycle by addition of fresh media containing nitrogen, as well as thiamine to maintain transcriptional repression of dpb2⁺. Cells were collected every 30 min for 6 h and the DNA content analyzed by flow cytometry. The positions of 1C and 2C DNA content are indicated.

Figure 4.

Temperature-sensitive mutant dpb2-d1 arrests in late G1-early S phase after shift to the restrictive temperature. (A) Amino acid sequence alignment of the most highly conserved region (motif III) of the putative phosphatase domain with a protein super family consisting of the B subunits of eukaryotic DNA polymerases and selected members of the regulatory subunits of the archael X-family DNA polymerases. The location of the conserved domains (motif I-IV) is shown for S. pombe Dpb2. Asterisks indicate the amino acids 413–417, deleted in the mutant dpb2-d1. Identical residues are shown in black. Conservative changes are indicated in gray. Species abbreviations are as follows: Af, Archaeoglobus fulgidus; Hs, Homo sapiens; Mj, Methanococcus jannaschii; Mm, Mus musculus; Mta, Methanothermobacter thermoautotrophicum; Pt, Pyrococcus furiosus; Sc, S. cerevisiae; and Sp, S. pombe. (B) Cells carrying wild-type dpb2⁺ or dpb2-d1 (Δ413–417) were streaked on minimal medium and incubated at 25°C or 36°C. Plates were photographed after 4 d. (C) Cell number analysis in dpb2-d1 cells after shift to the restrictive temperature. Wild-type 972 cells or dpb2-d1 (Δ413–417) were grown at 25°C in minimal medium to mid-log phase and shifted to 36°C for 8 h. Please note that after shift to the restrictive temperature, dpb2-d1 is limited to one population doubling (typical of an early S phase arrest), whereas the wild-type culture continues to grow exponentially. (D) dpb2-d1 cells arrest with a 1C DNA content after shift to restrictive temperature. dpb2⁺ (top) and dpb2-d1 (bottom) were grown at 25°C overnight. The following day, cells were shifted to the restrictive temperature of 36°C, collected at the indicated times, and measured for DNA content by flow cytometry. The position of 1C and 2C DNA content are indicated.

Figure 5.

4xFLAG-Dpb2 binds ARS elements early in S phase. (A) Analysis of DNA content by flow cytometry indicates that DNA synthesis begins at 60–80 min after release from the G2 block. (B) Relative enrichment ratio of immunoprecipitated ARS DNA to nonARS DNA was calculated and plotted from the raw data in C. (C and D) ChIP analyses with antibody to the FLAG-epitope for Dpb2 (C) or antibody to Mcm6 (D) were used to measure the levels of Dpb2 and Mcm6 at ARS loci. DNA isolated from the immunoprecipitated chromatin (IP) or from whole cell soluble extracts (Total DNA) was subjected to PCR to amplify DNA fragments from either ars2004, ars3002 or from nonARS DNA. Please note that the peak of Dpb2 binding to ARS elements occurs 60 min after release from the G2 block (C) in contrast to Mcm6 binding which peaks at 40 min postrelease (D). No significant chromatin binding was observed when cross-linking agent was omitted, if cell lysates were prepared in the absence of FLAG-tagged protein, or if antibodies were excluded from the immunoprecipitation reaction (our unpublished data).

Figure 6.

Dpb2 fails to associate with origin DNA in cdc10-129 cells at 36°C. (A) Exponentially growing 972 cells (lane 1), G2 arrested GD254 cells (lane 2), or G1 arrested GD261 cells (lanes 3 and 4) were subjected to ChIP analysis for the presence of chromatin bound Dpb2. Either magnetic beads conjugated to anti-FLAG antibodies (lanes 1, 2 and 4) or magnetic beads alone (lane 3) were used for immunoprecipitation. The relative ratio of immunoprecipitated DNA to total DNA was plotted. No enrichment of Dpb2 binding to ars2004 is observed in cdc10-129 cells arrested in G1 (lane 4). The levels of binding to either non-ARS or ARS-associated chromatin in cdc10 cells is equivalent to the level detected in either G2 arrested (lane 2) or exponentially growing cells (lane 1). Very little chromatin is precipitated from cdc10 cells in the absence of antibodies (lane 3). (B) ChIP analysis using anti-FLAG antibodies for detection of Pol ε-Cterm at ars2004.