The function of DNA polymerase alpha at telomeric G tails is important for telomere homeostasis

Abstract

Telomere length control is influenced by several factors, including telomerase, the components of telomeric chromatin structure, and the conventional replication machinery. Although known components of the replication machinery can influence telomere length equilibrium, little is known about why mutations in certain replication proteins cause dramatic telomere lengthening. To investigate the cause of telomere elongation in cdc17/pol1 (DNA polymerase alpha) mutants, we examined telomeric chromatin, as measured by its ability to repress transcription on telomere-proximal genes, and telomeric DNA end structures in pol1-17 mutants. pol1-17 mutants with elongated telomeres show a dramatic loss of the repression of telomere-proximal genes, or telomeric silencing. In addition, cdc17/pol1 mutants grown under telomere-elongating conditions exhibit significant increases in single-stranded character in telomeric DNA but not at internal sequences. The single strandedness is manifested as a terminal extension of the G-rich strand (G tails) that can occur independently of telomerase, suggesting that cdc17/pol1 mutants exhibit defects in telomeric lagging-strand synthesis. Interestingly, the loss of telomeric silencing and the increase in the sizes of the G tails at the telomeres temporally coincide and occur before any detectable telomere lengthening is observed. Moreover, the G tails observed in cdc17/pol1 mutants incubated at the semipermissive temperature appear only when the cells pass through S phase and are processed by the time cells reach G(1). These results suggest that lagging-strand synthesis is coordinated with telomerase-mediated telomere maintenance to ensure proper telomere length control.

FIG. 1

Loss of silencing in pol1-17 mutants does not occur at HML. Total RNA was isolated from strains CH2494 (MATaPOL1) at 24 and 30°C, CH2493 [MATapol1-17(short)], and CH2493 [MATapol1-17(long)]. Expression of MATα information from the HML locus was examined by Northern hybridization analysis of MATa strains with a probe that recognizes both the α1 and α2 transcripts, which comigrate on an agarose gel. Expression of the α1 and α2 transcripts is repressed to the same extent in POL1 cells (lanes 2 and 4), pol1-17(short) cells (lane 1), and pol1-17(long) cells (lane 3). As a positive control, RNA also was isolated from strain CH557 (MATα POL1) (lane 5). To monitor RNA loading in each lane, actin mRNA levels were measured by hybridization with an ACT1 probe. A longer exposure of this gel also fails to show any α1 or α2 transcripts in pol1-17(long) cells (lane 3).

FIG. 2

A telomere-proximal URA3 gene is rapidly derepressed in pol1-17 mutants upon the shift to 30°C. (A) Telomeric URA3 expression was examined in strains CH2514 [pol1-17(short)] (lane 1), CH2514 [pol1-17(long)] (lane 2), and CH2515 (POL1) grown at either 24°C (lane 3) or 30°C (lane 4). pol1-17(long) cells exhibit a dramatic loss of telomeric URA3 repression. (B) Strains CH2514 [pol1-17(short)] and CH2515 (POL1) were grown to early log phase at 24°C, and aliquots were shifted to 24 or 30°C for 2 or 5 h. URA3 derepression in pol1-17 cells occurs by 2 to 5 h after the shift to 30°C (compare lane 1 with lanes 4 and 5). For each sample in panels A and B, RNA was isolated and the expression of a telomere-proximal URA3 gene was examined by Northern hybridization with a URA3 probe. To monitor RNA loading in each lane, actin transcript levels were measured by hybridization with an ACT1 probe.

FIG. 3

pol1-17 mutants quickly exhibit single-stranded character at the telomeres upon the shift to 30°C. Strains CH2377 (pol1-17) and CH2378 (POL1) were grown to early log phase at 23°C, and aliquots were shifted to 30°C for various times. DNA isolated at each time point was digested with XhoI, which releases terminal telomeric fragments on Y′-containing telomeres, and analyzed by nondenaturing in-gel hybridization (A) and denaturing hybridization (B) with a CA oligonucleotide probe. Hybridization to high-molecular-weight DNA represents non-Y′-containing telomeres. ssDNA on the native gel is observed by 1 h after the shift to 30°C (lane 2), and its level increases by 2 h after the shift to 30°C (lane 3). Stripping and reprobing the gel under denaturing conditions demonstrates that the lanes were approximately evenly loaded. Lanes 15 and 16 contain control double-stranded and single-stranded TG_1–3 DNA, respectively. The smear of Y′-containing telomeric DNA from pol1-17 cells is indicated by an arrow in panel B. Molecular weight markers (in thousands) are indicated on the left.

FIG. 4

The increase in the level of telomeric ssDNA in pol1-17 cells is reversible. Strain CH2377 [pol1-17(short)], grown to early log phase at 23°C, was shifted to 30°C, and aliquots of cells were collected after 1 and 5 h of growth. Subsequently, the culture of pol1-17 cells was shifted back down to 23°C and aliquots again were collected after 1 and 5 h of growth. DNA isolated at each time point was digested with XhoI and analyzed as described in the legend to Fig. 3. (A) Nondenaturing in-gel hybridization with a CA-oligonucleotide probe reveals that the ssDNA that appears at 30°C (lanes 2 and 3) is lost immediately after the cells are shifted back to 23°C (lanes 4 and 5). (B) Stripping, denaturing, and reprobing of the native gel in panel A with a Y′ probe indicates that the lanes are approximately evenly loaded. Lanes 6, 7, and 8 contain control double-stranded TG_1–3 DNA, single-stranded TG_1–3 DNA, and Y′ DNA, respectively. Because a Y′ probe was used for this hybridization, the pattern of bands after denaturation differs in appearance from those in other figures. The smear of Y′-containing telomeric DNA is indicated by an arrow in panel B. Molecular weight (MW) markers (in thousands) are indicated in the middle.

FIG. 5

Telomeric ssDNA in pol1-17 cells is removed by ExoI. CH2377 [pol1-17(short)] was grown to early log phase at 23°C and shifted to 30°C for 5 h. DNA was isolated before and after the temperature shift and incubated in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of ExoI. The DNA was digested with XhoI and examined by nondenaturing in-gel hybridization (A) and denaturing hybridization (B) with a CA-oligonucleotide probe, as described in the legend to Fig. 3. Telomeric ssDNA in pol1-17 cells is lost following treatment with ExoI (compare lanes 3 and 4 in panel A). Lanes 5 and 6 contain control single-stranded and double-stranded TG_1–3 DNA, respectively. Molecular weight (MW) markers (in thousands) are indicated in the middle.

FIG. 6

ssDNA tails occur in the absence of telomerase, but the telomeres do not elongate. DNA was isolated from strain RWY120 (cdc17-1 tlc1Δ) cells or from strain RWY120 cells carrying a plasmid-borne copy of TLC1 after growth at 23°C (lanes 1 and 5) and after the 23°C culture had been shifted to 30°C for 2 h (lanes 2 and 6), 6 h (lanes 3 and 7), or 24 h (lanes 4 and 8) of growth. (A) Nondenaturing in-gel hybridization with a CA-oligonucleotide probe indicates that the telomeric ssDNA that is present in cdc17-1 cells at 30°C (lanes 2 to 4) is produced even in the absence of telomerase (lanes 6 to 8). (B) Denaturing hybridization with a CA-oligonucleotide probe of the gel in panel A demonstrates that the lanes are approximately evenly loaded. Note that the telomeres are substantially shorter in the cdc17-1 tlc1Δ strain than in the cdc17-1 strain; telomere shortening occurs during the 30 generations of growth at 23°C that is necessary to produce the cdc17-1 tlc1Δ strain (compare telomeric DNA smears [arrow]).

FIG. 7

The appearance of telomeric ssDNA occurs during S phase in pol1-17 cells at 30°C. (A) In-gel hybridization of DNA derived from pol1-17 cells arrested in G₁ (lanes 1 to 3) and released into a synchronous cell cycle (lanes 4 to 8) at 23°C (left panels) and 30°C (right panels). Exponentially growing cells of strain RWY121 (pol1-17 bar1Δ) were treated with α-factor for 11 h at 23°C to arrest the cells in the G₁ phase. The culture was then split into two portions, which were incubated at 23 or 30°C for the remainder of the experiment. The portions were split into two aliquots after 1 h (t = 0, lanes 1), and the cells of one aliquot were kept in G₁ with α-factor (lanes 2 and 3) while the other aliquot was released into the cell cycle by α-factor removal (lanes 4 to 8). Samples of cells were collected from all four aliquots at 0.5 h (lanes 2 and 4), 1 h (lanes 5), 2 h (lanes 6), 3 h (lanes 7), and 5 h (lanes 3 and 8) after t = 0. These aliquots were used for DNA isolation and analysis of the DNA end structures by nondenaturing in-gel hybridization (as described in the legend to Fig. 3) (A); they also were used for FACS analysis of DNA content (B). Hybridization under denaturing conditions demonstrates that all lanes were approximately evenly loaded (lower panels in panel A). Lanes 9 and 10 contain control single-stranded and double-stranded TG_1–3 DNA, respectively. Note that for the cultures remaining in G₁, only the DNA isolated after 0.5 and 5 h is shown. The DNA isolated from cells harvested at the time points in between those two times yielded indistinguishable very weak hybridization in the nondenaturing gels (data not shown). Similarly, only the FACS profiles of cells at t = 0 and cells collected after the release into the cell cycle are shown; the profiles of the cells remaining at G₁ were indistinguishable from those at t = 0 (data not shown). pol1-17 cells released into the cell cycle at 30°C display an excess of ssDNA that initially appears only when the cells enter S phase. Molecular weight (M) markers (in thousands) are indicated on the left of each panel.

FIG. 8

The telomeric ssDNA structures generated in S phase can be processed to normal end structures in pol1-17-cells even at 30°C. Strain RWY121 (pol1-17 bar1Δ) was grown exponentially at 23°C and then shifted to 30°C overnight to allow production of telomeric ssDNA (0h, lane 1). The 30°C culture was then arrested in G₁ with 600 μM α-factor for 2 h (lane 2) or 6 h (lane 3). (A) Nondenaturing in-gel hybridization to DNA derived from the cells at the individual time points (top panel) and the same gel hybridized after denaturing the DNA (bottom panel) as in Fig. 3. (B) FACS profiles of the same cells used in panel A. Note that pol1-17 cells growing at the semipermissive temperature always show a predominance of cells with a 2C DNA content (1). Cell- cycle arrest in the G₁ phase of this culture was also monitored by measuring cell morphology (shmooed and single cells versus budded cells) at each time point to confirm that >85% of the cells were arrested in G₁ after 6 h of α-factor treatment (data not shown). The ssDNA tails generated in pol1-17 cells at 30°C are processed to normal end structures by the time the cells reach the G₁ phase. Molecular weight (M) markers (in thousands) are indicated on the left of each panel.