Schizosaccharomyces pombe cells lacking the amino-terminal catalytic domains of DNA polymerase epsilon are viable but require the DNA damage checkpoint control

Abstract

In Schizosaccharomyces pombe, the catalytic subunit of DNA polymerase epsilon (Pol epsilon) is encoded by cdc20(+) and is essential for chromosomal DNA replication. Here we demonstrate that the N-terminal half of Pol epsilon that includes the highly conserved polymerase and exonuclease domains is dispensable for cell viability, similar to observations made with regard to Saccharomyces cerevisiae. However, unlike budding yeast, we find that fission yeast cells lacking the N terminus of Pol epsilon (cdc20(DeltaN-term)) are hypersensitive to DNA-damaging agents and have a cell cycle delay. Moreover, the viability of cdc20(DeltaN-term) cells is dependent on expression of rad3(+), hus1(+), and chk1(+), three genes essential for the DNA damage checkpoint control. These data suggest that in the absence of the N terminus of Pol epsilon, cells accumulate DNA damage that must be repaired prior to mitosis. Our observation that S phase occurs more slowly for cdc20(DeltaN-term) cells suggests that DNA damage might result from defects in DNA synthesis. We hypothesize that the C-terminal half of Pol epsilon is required for assembly of the replicative complex at the onset of S phase. This unique and essential function of the C terminus is preserved in the absence of the N-terminal catalytic domains, suggesting that the C terminus can interact with and recruit other DNA polymerases to the site of initiation.

FIG. 1

Schematic representation of N-terminally truncated forms of cdc20 that were used for the complementation analysis shown in Fig. 2 (the gene structure is not drawn in scale). All plasmids were derived from pIRT2-cdc20⁺, which contains a 10,054-bp genomic fragment of the cdc20⁺ gene. The numbers in parentheses indicate nucleotide positions. The first nucleotide of the start codon was numbered 1, and all other sequences were designated accordingly. The numbers on the right of each plasmid indicate the numbers of amino acids of Pol ɛ being included in each construct. The series of pRep1 plasmids with which the C-terminal fragments of the cdc20 product were expressed under the nmt1 promoter were generated by cloning restriction fragments of pIRT2-cdc20⁺ into the pRep1 vector. Initiation of translation is presumed to take place at the first internal methionine. The putative NLS and zinc finger motifs are as indicated.

FIG. 2

Expression of the C-terminal half of Pol ɛ rescues both cdc20^ts mutants and cells with the complete cdc20⁺ gene deleted. (A) Complementation of the temperature-sensitive cdc20-M10 (top) and cdc20-P7 (bottom) strains by transformation with plasmids expressing the C-terminally truncated forms of Pol ɛ. Transformants were streaked on minimal agar and incubated at 25°C (left) and 36°C (right). (B) Expression of the C-terminal half of the cdc20 product can rescue the Δcdc20 strain. The cdc20⁺/Δcdc20 diploid strain was transformed with pIRT2-cdc20⁺ or pIRT2-cdc20C1. Following sporulation and germination of positive transformants, haploid cells containing the deletion of cdc20⁺ and either the plasmid pIRT2-cdc20⁺ (top) or pIRT2-cdc20C1 (bottom) were selected and visualized by phase contrast microscopy.

FIG. 3

Construction of the cdc20^ΔN-term mutant. (A) Expected genomic structure of the cdc20 and leu1 loci following integration of pJK148-cdc20C1. The solid and cross-hatched bars indicate the regions of cdc20 corresponding to the N terminus and the C terminus, respectively. P indicates the location of the PstI restriction sites used for the Southern blot analysis. (B) Southern blot of genomic DNA prepared from the diploid Δcdc20/cdc20⁺ strain (lane 3) and from two independent cdc20^ΔN-term isolates (lanes 1 and 2), probed with a PCR fragment corresponding to the C-terminal half encoded by cdc20. (C) Western blot of a protein extract prepared from wild-type cells (lane 1) and cdc20^ΔN-term cells (lane 2), using anti-HA monoclonal antibodies. The apparent molecular mass of 3HACdc20C1p is approximately 122 kDa.

FIG. 4

Cells lacking the N-terminal half of Pol ɛ display increased sensitivity to DNA damage. Survival rates of cdc20^3hacdc20+ (triangle), cdc20^ΔN-term (square), cdc20^{N-term+C-term} (circle), hus1-14 (diamond in panel A), and rad2-44 (diamonds in panels B and C) cells. Following treatment with 11 mM HU (A), increasing doses of UV irradiation (B), and 0.2% MMS (C), cells were plated at 32°C for 3 days, colonies were counted, and the survival rate was determined by SigmaPlot. Error bars indicate standard deviations.

FIG. 5

Cells lacking the N-terminal half of Pol ɛ show delayed S and G₂ phases of the cell cycle. (A) FACS analysis of the DNA content of cells released from the cdc10-129 cell cycle arrest. Samples were collected every 15 min for approximately 2 h. The 1C and 2C DNA control peaks are indicated. (B) Percentage of binucleate cells for cdc10-129 cdc20^3hacdc20+ (square), cdc10-129 cdc20^ΔN-term (circle), and cdc10-129 cdc20^{N-term+C-term} (diamond) strains at the indicated times following release from the G₁ block.

FIG. 6

Cell viability for the cdc20^ΔN-term strain is dependent on the DNA damage checkpoint control. The cdc20^tsN-term^+C-term Δchk1 double mutant is inviable at 36°C. (A) Exponentially growing cultures of 972⁺ (square), cdc20^{N-term+C-term} (diamond), cdc20^tsN-term^+C-term (circle), cdc20^{N-term+C-term} Δchk1 (triangle), and cdc20^{tsN-term+C-term} Δchk1 (closed circle) strains were shifted from 25 to 36°C for 10 h. Samples were collected every hour and plated to determine cell viability. (B) The loss of viability of cdc20^{tsN-term+C-term}Δchk1 cells at 36°C correlates with an increase in the number of cells undergoing an aberrant mitosis. These events are plotted as the percentage of “cut” cells and cells with abnormal nuclear morphology. (C) Visualization of the cut phenotype by DAPI staining of nuclei. Panels 1 and 2, cdc20^{N-term+C-term} Δchk1 cells at 25 and 36°C, respectively. Panels 3 and 4, cdc20^{tsN-term+C-term} Δchk1 cells at 25 and 36°C, respectively.