The Ctf18RFC Clamp Loader Is Essential for Telomere Stability in Telomerase-Negative and <i>mre11</i> Mutant Alleles

<div><p>The function of the replication clamp loaders in the semi-conservative telomere replication and their relationship to telomerase- and recombination mechanisms of telomere addition remains ambiguous. We have investigated the variant clamp loader Ctf18 RFC (Replication Factor C). To understand the role of Ctf18 at the telomere, we first investigated genetic interactions after loss of Ctf18 and TLC1 (the yeast telomerase RNA). We find that the <i>tlc1▵ ctf18▵</i> double mutant confers a rapid >1000-fold decrease in viability. The rate of loss was similar to the kinetics of cell death in <i>rad52▵ tlc1▵</i> cells. However, the Ctf18 pathway is distinct from Rad52, required for the repair of DSBs, as demonstrated by the synthetic lethality of <i>rad52▵ tlc1▵ ctf18▵</i> triple mutants. These data suggest that each mutant elicits non-redundant defects acting on the same substrate. Second, interactions of the yeast hyper-recombinational mutant, <i>mre11A470T, with ctf18▵</i> confer a synergistic cold sensitivity. The phenotype of these double mutants ultimately results in telomere loss and the generation of recombinational survivors. We observed a similar synergism between single mutants that led to hypersensitivity to the DNA alkylating agent, methane methyl sulphonate (MMS), the replication fork inhibitor hydroxyurea (HU), and to a failure to separate telomeres of sister chromatids. Hence, <i>ctf18▵</i> and <i>mre11A470T</i> act in different pathways on telomere substrates for multiple phenotypes. The <i>mre11A470T</i> cells also displayed a DNA damage response (DDR) at 15°C but not at 30°C while <i>ctf18▵</i> mutants conferred a constitutive DDR activity. Both the 15°C DDR pattern and growth rate were reversible at 30°C and displayed telomerase activity <i>in vivo</i>. We hypothesize that Ctf18 confers protection against stalling and/or breaks at the replication fork in cells that either lack, or are compromised for, telomerase activity. This Ctf18-based function is likely to contribute another level to telomere size homeostasis.</p></div

Western blot analysis of DNA damage responses to <i>mre11A470T</i> and <i>ctf18</i>▵ Mutations.

<p>[<b>Top</b>]: Protein extracts were isolated from wild-type, <i>mre11A470T</i>, <i>ctf18▵</i> and <i>mre11A470T ctf18▵</i> cultures grown in 5 ml YPD cultures at 30°C or 15°C. Extracts were subjected to electrophoresis on a 10% SDS-PAGE gel prior to Western analysis using goat primary polyclonal anti-Rad53 antibody as described in Materials and Methods. Phosphorylated forms (arrows on right) migrate more slowly than the un-phosphorylated species. [<b>Bottom</b>]: Protein extracts were isolated from cold resistant <i>mre11A470T ctf18Δ</i> survivors and grown in 5 ml YPD at 15°C before shifting to 30°C. Extracts were subjected to Western analysis for Rad53 expression as described above.</p

<i>mre11A470T ctf18</i>▵ cold sensitive survivors arise via recombinational mechanisms.

<p>[<b>GT</b>] DNA isolated from the indicated strains (top) grown at 30°C and 15°C and from ten independent survivors (1–10) were digested with XhoI and Southern analysis performed using poly GT as a telomeric probe. Arrow on right indicates position of Type I survivor. Note that the smearing of the distribution that represents the distribution of Type II-like survivors. [<b>PEP4</b>] Southern blot from above was stripped and probed with <i>PEP4</i> sequences as an internal loading control.</p

Senescence of telomerase-negative strains in the presence and absence of Ctf18.

<p>[<b>A</b>]: Senescence assay growth curve generated during 196 (11 rounds) of repetitive subculturing for wild type, <i>mre11A470Ttlc1▵</i>, <i>ctf18▵</i> and <i>mre11A470T ctf18▵</i> strains. Colonies from freshly dissected spores were incubated on YPD plates for 18 hours at 30°C and then inoculated into 5 ml YPD, grown at 30°C for 18 hours [36 hours after dissection], and counted using a hemocytometer. Each subsequent round of subculturing [hours 54–198] was initiated by inoculating fresh medium with 1×10⁵ cells/ml from the previous subculturing at 30°C. [<b>B</b>]: Telomere lengths of the strains in [1A] after 36 and 54 hours of growth after dissection at 30°C. The samples shown on the gel were derived from the cells grown in [<b>1A</b>]. Telomere sizes were determined by digestion of genomic DNA with XhoI followed by Southern analysis using poly GT as a telomeric probe.</p

<i>mre11A470T pol32 Δ</i> is cold sensitive for growth.

<p>Ten microliters of wild type, <i>mre11A470T</i>, <i>pol32▵</i> and <i>mre11A470T pol32▵</i> cultures grown at 30°C were spotted onto YPD plates at 10-fold serial dilutions [arrow above] and incubated at either 30°C [left] for two days or at 15°C [right] for five days of growth.</p

<i>mre11A470T ctf18Δ</i> cells have sister telomere separation defects.

<p>Late anaphase cells identified from asynchronous cultures of wild-type, and <i>mre11A470T ctf18▵</i> strains grown at 30°C and characterized by fluorescence microscopy to visualize GFP signals as described in Materials and Methods. Telomeres on chromosome IV-L were labeled with GFP-LacI fusion proteins bound to LacO arrays adjacent to telomeres. The mitotic spindles were labeled with the <i>TUB1</i>-GFP fusion protein. The left panel displays the bright field image while the right panel the fluorescent signal from the same field.</p

<i>mre11A470T ctf18Δ</i> is cold sensitive for growth.

<p>[Top] Ten microliters of wild type, <i>mre11A470T</i>, <i>ctf18▵</i> and <i>mre11A470T ctf18▵</i> cultures grown at 30°C were spotted onto YPD plates at 10-fold serial dilutions [arrow] and incubated at either 30°C [left] for two days or at 15°C [right] for five days to compensate for the lower growth rate of the latter. [Bottom] Cells of the indicated strains were also transformed with the <i>CTF18</i>-containing plasmid p5472 were spotted onto uracil omission media at 10-fold serial dilutions. Plates were then incubated at 15°C for fourteen days.</p

<i>mre11A470T ctf18Δ</i> cells are sensitive to replicative inhibitors and alkylating mutations.

<p>Wild type, <i>mre11A470T</i>, <i>ctf18Δ</i> and <i>mre11A470T ctf18Δ</i> cultures grown at 30°C were spotted onto YPAD in 10-fold serial dilutions [arrow on top] without [left] or with [right] 40 mM of the replicative inhibitor hydroxyurea [HU] and incubated at 30°C. The same cells were treated in the presence or absence of 0.0005% methane methyl sulphonate (MMS) and incubated at 30°C.</p

<i>rad52▵ tlc1▵ ctf18▵</i> haploid strain requires <i>RAD52</i> for viability.

<p>[<b>A</b>]: The CP1 diploid heterozygous for <i>tcl1</i>::<i>LEU2</i>, <i>ctf18</i>::<i>KanMX</i>, and <i>rad52</i>::<i>TRP1</i> were transformed with the <i>RAD52</i>-containing <i>CEN</i> plasmid, pWJ1213, giving rise to CP2. CP2 cells were grown on solid YPD medium for 72 hours to allow for plasmid loss. Single colonies were then patched onto both histidine-omission media and YPD to determine the rate of plasmid loss. [<b>B</b>]: Products of random spore analysis of CP2 grown on YPD for 72 hours in an attempt to isolate a viable haploid <i>tcl1</i>::<i>LEU2</i>, <i>ctf18</i>::<i>KanMX</i>, and <i>rad52</i>::<i>TRP1</i> strain. Of the 75 triple mutant haploid spores tested, none were able to lose the plasmid, as evidenced by continued growth on histidine-omission media, and retention of growth on YPD.</p

Senescence of telomerase-negative strains in the presence and absence of Rad52.

<p>[<b>A</b>]: Cells were subcultured for eleven rounds (198 generations post germination) of subculturing for the indicated strains as described above. Colonies from freshly dissected spores were incubated on YPD plates for 36 hours at 30°C, and 1×10⁵ cells were inoculated into 5 ml of YPD at 30°C for 18 hours [54 generations after dissection], and cells counted using a hemocytometer. Each subsequent 18-hour round of grown [hours 72–198] was initiated by inoculating 1×10⁵ cells into fresh YPD. [<b>B</b>]: Telomere lengths of the indicated strains in [<b>2A</b>] from hours 54, 72 and 90 hours after dissection were determined by Southern analysis of XhoI digested DNA using poly GT as a probe.</p