Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events - PubMed (original) (raw)

Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events

Q Chen et al. Mol Cell Biol. 2001 Mar.

Abstract

Yeast cells can survive in the absence of telomerase RNA, TLC1, by recombination-mediated telomere elongation. Two types of survivors, type I and type II, can be distinguished by their characteristic telomere patterns. RAD52 is essential for the generation of both types of survivors. Deletion of both RAD50 and RAD51 produces a phenotype similar to that produced by deletion of RAD52. Here we examined the effects of the RAD50 and the RAD51 epistasis groups as well as the RAD52 homologue, RAD59, on the types of survivors generated in the absence of telomerase. rad59 mutations completely abolished the ability to generate type II survivors, while rad50 mutations decreased the growth viability of type II survivors but did not completely eliminate their appearance. Mutations in RAD51, RAD54, and RAD57 had the converse affect: they eliminated the ability of cells to generate type I survivors in a tlc1 strain. The triple mutant, tlc1 rad51 rad59, was not able to generate survivors. Thus either type I or type II recombination pathways can allow cells to survive in the absence of telomerase; however, elimination of both pathways in a telomerase mutant leads to the inability to elongate telomeres and ultimately cell death.

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Figures

FIG. 1

RAD59 is required to generate survivors in a rad51 background. Cells were grown to saturation in YPD medium and then diluted to 105 cells/ml every 24 h with fresh YPD medium. Cells were counted every 24 h with a hemocytometer. The curves shown are the average of results for four independent clones from each genotype.

FIG. 2

The tlc1 rad51 rad59 mutant does not show a higher rate of telomere shortening. tlc1, tlc1 rad52, and tlc1 rad51 rad59 cells were grown for five successive days in liquid culture, and telomere length was determined on Southern blots for the first 3 days. The tlc1 rad52 and tlc1 rad51 rad59 mutants reached a minimum growth rate at day 4. Solid arrows indicate X telomeres and the open arrow indicates Y′ telomeres in the wild-type strain.

FIG. 3

tlc1 rad59 double mutants generate only type I survivors. Southern blots of tlc1 and tlc1 rad59 mutants are shown. The numbers at the top of the lane represent the number of days that cells were grown in liquid culture. The letter S at the top of the lanes represents survivor cells that were streaked out at least two times after survivors were generated. The numbers on the side indicate the DNA molecular size markers in kilobases. The survivor type is indicated below each lane as type I or type II. Solid arrows indicate X telomeres and open arrows indicate Y′ telomeres in the wild-type strain. (A) tlc1 mutants were grown in liquid culture, and telomeres were measured on Southern blots at days 1 and 4, before the generation of survivors (lanes 1 and 2). At days 9, 10, and 15, type II survivors were apparent (lanes 3 to 5). Wild-type telomeres are shown for comparison in lane 6. (B) The telomere patterns of tlc1 rad59 double mutants are shown before the generation of survivors at days 1, 4, and 9 (lanes 2 to 4) and after type I survivors were generated at days 13, 17, and 21 (lanes 5 to 7). (C) The single-colony assay was used for tlc1 rad59 cells. Wild-type cells (lane 1), a presurvivor colony at the second streak-out (lane 2), and four independent type I tlc1 rad59 survivor colonies are shown.

FIG. 4

Type II survivors that are generated in tlc1 rad50 cells are not maintained during continuous culturing. tlc1 cells were cultured for 15 days, and telomeres were examined at days 1, 4, 9, 10, and 15 (lanes 1 to 5). Type II survivors were detected at days 9, 10, and 15 (lanes 3 to 5). tlc1 rad50 cells were grown for 23 days, and telomeres were examined at days 1, 4, 10, 15, 19, and 23 (lanes 8 to 12). Type II survivors were detected at day 10 but not at days 15, 19, and 23. The survivor type is indicated below each lane as type I or type II.

FIG. 5

tlc1 rad51 mutants generate only type II survivors in the single-colony assay. tlc1 and tlc1 rad51 cells were streaked out repeatedly until survivors were generated (see Material and Methods). DNA samples from either presenescence cells (streak 2) or survivor cells (S) were used for telomere length pattern analysis. (A) For tlc1, both type I (lanes 3 to 5) and type II (lanes 2 and 6) survivors were detected. (B) For tlc1 rad51, of four independent survivors, all were type II (lanes 3 to 6). (C) For tlc1 rad54, of four independent survivors, all were type II (lanes 3 to 6). (D) For tlc1 rad57, of four independent survivors, all were type II (lanes 3 to 6). The survivor type is indicated below each lane. Solid arrows indicate X telomeres and open arrows indicate Y′ telomeres in the wild-type strain.

FIG. 6

Reintroduction of the RAD59 gene into late-generation tlc1 rad59 survivor cultures does not reestablish a wild-type distribution of survivor types. (A) The pRAD59 plasmid was transformed into tlc1 rad59 cells at various times during the generation of survivor cells as indicated. (B) tlc1 rad59 spore clones that did not contain the pRAD59 plasmid (lanes 2 to 6) and those that did (lanes 7 to 11) were analyzed after 12 days of growth in liquid culture. Two additional spore clones containing the pRAD59 plasmid were also analyzed at day 12. (C) pRAD59 was transformed into tlc1 rad59 cells at generation ∼60, and independent transformants were examined for their survivor type distribution by Southern blot analysis. Two independent transformants containing pRAD59 (lanes 2 to 4 and 5 to 7) were examined at days 1, 5, and 8. Control cells transformed with the pRS315 vector only are shown in lane 8. Additional independent transformants containing the pRAD59 plasmid were assayed at day 8 (lanes 9 to 13) or day 11 (lane 14). (D) pRAD59 or the vector pRS315 was transformed into tlc1 rad59 cells at generation ∼130 (see Material and Methods), and independent transformants were cultured for 4 days to test survivor types. Two transformants containing pRAD59 (lanes 2 to 5 and 6 to 9) and one with the pRS315 vector (lanes 10 to 13) were assayed for telomere pattern. Additional independent colonies containing pRAD59 (lanes 15 to 18) or vector (lane 19) at day 4 were assayed. Numbers on the side are molecular size markers in kilobases. Lanes marked wt contain wild-type DNA as a control. Plas, plasmid.

FIG. 7

Two types of survivors are generated by two distinct genetic pathways. A telomere containing two tandem Y′ elements (large gray boxes) separated by TG1–3 repeats (small white boxes) is represented at the top. Telomere shortening occurs in the absence of telomerase. Survivors can be generated via two different mechanisms. (A) The _RAD51_-, _RAD54_-, and _RAD57_-dependent pathway generates type I survivors. Telomere shortening continues into the Y′ element, exposing single-stranded 3′ overhangs. The single-stranded DNA invades a homologous region in the Y′ element on some other telomere, and BIR allows duplication of the intact telomere onto the telomere which had lost the TG1–3 repeats. The telomere that is copied is shown in light gray for clarity, to reveal the 3′ end elongation of the invading strand. This kind of recombination event could also occur at X sequences on telomeres that do not contain Y′ elements (see the text). (B) Telomere shortening results in recombination before all of the telomere repeats are lost from the ends. RAD50 and RAD59 allow recombination in the irregular TG1–3 repeats to occur efficiently. Alternatively, the telomeric TG1–3 tracts self-prime DNA replication and allow extension of the telomere sequences with a rolling-circle-type mechanism.

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Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events - PubMed (original) (raw)