Involvement of replicative polymerases, Tel1p, Mec1p, Cdc13p, and the Ku complex in telomere-telomere recombination

Abstract

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of the reverse transcriptase telomerase. In both tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. Genetic studies have led to the identification of DNA polymerases, cell cycle checkpoint proteins, and telomere binding proteins involved in the telomerase pathway. However, how these proteins affect telomere-telomere recombination has not been identified to date. Using an assay to trace the in vivo recombinational products throughout the course of survivor development, we show here that three major replicative polymerases, alpha, delta, and epsilon, play roles in telomere-telomere recombination and that each causes different effects and phenotypes when they as well as the telomerase are defective. Polymerase delta appears to be the main activity for telomere extension, since neither type I nor type II survivors arising via telomere-telomere recombination were seen in its absence. The frequency of type I versus type II is altered in the polymerase alpha and epsilon mutants relative to the wild type. Each prefers to develop a particular type of survivor. Moreover, type II recombination is mediated by the cell cycle checkpoint proteins Tel1 and Mec1, and telomere-telomere recombination is regulated by telomere binding protein Cdc13 and the Ku complex. Together, our results suggest that coordination between DNA replication machinery, DNA damage signaling, DNA recombination machinery, and the telomere protein-DNA complex allows telomere recombination to repair telomeric ends in the absence of telomerase.

FIG. 1.

Replicative polymerases exhibit distinct phenotypes in telomere-telomere recombination. Liquid cultures were generated as described in Materials and Methods and diluted repeatedly 3:10,000 into fresh YEPD medium at 72 h at 30°C, the semipermissive temperature for these polymerase mutants. Genomic DNA from single mutants (A) and double mutants (B) from each dilution was digested with a combination of AluI, HaeIII, HinfI, and MspI, fractionated through 1% agarose, and transferred to a nylon filter. The filter was hybridized subsequently to a C_1-3A probe. An equal amount of DNA was loaded in each lane. An asterisk marks the position of critically short telomeres, and the arrow indicates the TG_1-3/C_1-3A fragments between Y′-Y′ tandem repeats. WT, wild type. Size markers (kilobases) are shown on the left.

FIG. 2.

Restriction analysis by XhoI digestion supports the involvement of replicative polymerases in telomere-telomere recombination. The experiment was conducted as described for Fig. 1, except that XhoI was used for restriction digestion. Size markers (kilobases) are shown on the left.

FIG. 3.

Accelerated senescent phenotype in telomerase-deficient and polymerase-defective mutants. (A) Cultures of wild-type (WT), tlc1, tlc1 cdc17-2, tlc1 cdc2-2, and tlc1 pol2-18 strains were inoculated from spores and grown to the stationary phase at 30°C. Fresh cultures were then inoculated to 2 × 10⁵ cells/ml and grown for 24 h, and cell numbers from the overnight cultures were determined and graphed. (B) Each strain was repeatedly streaked on solid YEPD plates and grown for 3 days at 30°C. It was noticeable that all telomerase-minus, polymerase-defective spores senesced faster, and survivors showed up much more seldom than those of the tlc1 mutant. (C and D) Analysis of telomeric pattern of single survivors. Survivors were generated from the solid-plate streaking. DNAs from each strain were prepared, and Southern blot analysis was performed as described for Fig. 1 and 2 by either four-base cutter (C) or XhoI (D) digestion. Three type I and three type II survivors from the tlc1 cdc17-2 and tlc1 pol2-18 mutants and six survivors from the tlc1 cdc2-2 mutant are shown here. Size markers (kilobases) are shown at the left.

FIG. 4.

Maintenance of tlc1 cdc2-2 survivors requires Rad52p. (A and B) Cultures of the tlc1 cdc2-2 strain were inoculated from spore colonies and grown to the stationary phase at 30°C. Stationary-phase cultures were diluted repeatedly 3:10,000 into fresh medium at 72 h as described for Fig. 1. The cultures were grown at 30°C for the first two dilutions and switched to 23°C, the permissive temperature, for the remaining dilutions. DNA was prepared for Southern analysis as described for Fig. 1A and 2B. Size markers (kilobases) are shown on the left. (C) Spores of rad52 tlc1 cdc2-2 and tlc1 cdc2-2 strains carrying a RAD52 URA3 plasmid were isolated on complete medium lacking uracil. Survivors were generated by restreaking spore colonies on YEPD plates. Shown here are two tlc1 cdc2-2 (left) and two rad52 tlc1 cdc2-2 (right) survivors without the RAD52 plasmid on a 5-fluorouracil (FOA) plate at 30°C. The plate is the first restreak from survivors on the YEPD plates. WT, wild type.

FIG. 5.

TEL1 and MEC1 genes are required for type II telomere-telomere recombination. Freshly dissected tlc1 (A), tlc1 tel1 (B), tlc1 mec1 sml1 (C), and tlc1 tel1 mec1 sml1 (D) spores were inoculated into YEPD medium. At 48-h intervals, DNA was isolated, and the cultures were then diluted 1:10,000 into fresh medium. This process was performed eight times. DNA was prepared from each time point for each strain and analyzed as for Fig. 1. Size markers (kilobases) are shown at the left.

FIG. 6.

Telomeric single-stranded binding protein Cdc13 influences survivor formation. (A) Liquid cultures and telomere Southern analysis were performed as described for Fig. 1 except that the cultures were grown at 27°C, the semipermissive temperature for the tlc1 cdc13-1 strain. Size markers (kilobases) are shown at the left. (B) The senescence and recovery rate of the wild-type (WT), tlc1, and tlc1 cdc13-1 strains were determined as described for Fig. 3A except that the cultures were grown at 27°C. (C) Growth of the tlc1 cdc13-1 strain on solid plates was examined by repeated streaking on YEPD plates and growth at 27°C.