Break-Induced Replication Requires DNA Damage-Induced Phosphorylation of Pif1 and Leads to Telomere Lengthening

Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR). Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway – Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening.     

Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping

Essential telomere 'capping' proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA <5 kb from the chromosome end, stimulating weak checkpoint activation; resection is extended >5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.     

Repair of DNA broken ends is similar in embryonic fibroblasts with and without telomerase

Telomeres cap the ends of chromosomes, preventing end-to-end fusions and subsequent chromosome instability. Here we used a telomerase knockout model to investigate whether telomerase participates in the processes of DNA break repair by de novo synthesis of telomere repeats at broken chromosome ends (chromosome healing). Chromosome healing giving rise to new detectable telomeric signals has not been observed in embryonic fibroblasts of telomerase-proficient mice exposed to ionizing radiation. Since the synthesis of telomeric sequences to broken DNA ends would make them refractory to rejoining events, the efficiency of rejoining of broken chromosomes in cell environments with and without telomerase has also been investigated. We conclude that the efficiency of rejoining broken chromosomes is not significantly different in the two cell environments. All together, our results indicate that there is no significant involvement of telomerase in the healing of broken DNA ends by synthesizing new telomeres in mouse embryo fibroblasts after exposure to ionizing radiation.     

NEJ1 prevents NHEJ-dependent telomere fusions in yeast without telomerase

In a search for genes involved in cell-type-dependent chromosome instability, we have found a role for NEJ1, a regulator of nonhomologous end joining (NHEJ), in cells that survive in the absence of telomerase. In yeast, NHEJ is regulated by mating-type status through NEJ1, which is repressed in a/alpha cells. For efficient NHEJ, NEJ1 is required as part of a complex with LIF1 and DNL4, which catalyzes DNA ligation. In haploid cells without telomerase, we find that the absence of NEJ1 results in high frequencies of circular chromosomes in type II survivors (i.e., those typified by lengthened telomere repeat tracts). These telomere fusion events are DNL4 dependent. NEJ1 therefore has a role in protecting telomeres from end fusions by NHEJ in the absence of telomerase that contrasts with its role in promoting repair at sites of DNA double-strand breaks.     

Chromosome instability as a result of double-strand breaks near telomeres in mouse embryonic stem cells

Telomeres are essential for protecting the ends of chromosomes and preventing chromosome fusion. Telomere loss has been proposed to play an important role in the chromosomal rearrangements associated with tumorigenesis. To determine the relationship between telomere loss and chromosome instability in mammalian cells, we investigated the events resulting from the introduction of a double-strand break near a telomere with I-SceI endonuclease in mouse embryonic stem cells. The inactivation of a selectable marker gene adjacent to a telomere as a result of the I-SceI-induced double-strand break involved either the addition of a telomere at the site of the break or the formation of inverted repeats and large tandem duplications on the end of the chromosome. Nucleotide sequence analysis demonstrated large deletions and little or no complementarity at the recombination sites involved in the formation of the inverted repeats. The formation of inverted repeats was followed by a period of chromosome instability, characterized by amplification of the subtelomeric region, translocation of chromosomal fragments onto the end of the chromosome, and the formation of dicentric chromosomes. Despite this heterogeneity, the rearranged chromosomes eventually acquired telomeres and were stable in most of the cells in the population at the time of analysis. Our observations are consistent with a model in which broken chromosomes that do not regain a telomere undergo sister chromatid fusion involving nonhomologous end joining. Sister chromatid fusion is followed by chromosome instability resulting from breakage-fusion-bridge cycles involving the sister chromatids and rearrangements with other chromosomes. This process results in highly rearranged chromosomes that eventually become stable through the addition of a telomere onto the broken end. We have observed similar events after spontaneous telomere loss in a human tumor cell line, suggesting that chromosome instability resulting from telomere loss plays a role in chromosomal rearrangements associated with tumor cell progression.     

Dynamic regulation of single-stranded telomeres in Saccharomyces cerevisiae

The temperature-sensitive phenotypes of yku70Delta and yku80Delta have provided a useful tool for understanding telomere homeostasis. Mutating the helicase domain of the telomerase inhibitor Pif1 resulted in the inactivation of cell cycle checkpoints and the subsequent rescue of temperature sensitivity of the yku70Delta strain. The inactivation of Pif1 in yku70Delta increased overall telomere length. However, the long G-rich, single-stranded overhangs at the telomeres, which are the major cause of temperature sensitivity, were slightly increased. Interestingly, the rescue of temperature sensitivity in strains having both pif1-m2 and yku70Delta mutations depended on the homologous recombination pathway. Furthermore, the BLM/WRN helicase yeast homolog Sgs1 exacerbated the temperature sensitivity of the yku70Delta strain. Therefore, the yKu70-80 heterodimer and telomerase maintain telomere size, and the helicase activity of Pif1 likely also helps to balance the overall size of telomeres and G-rich, single-stranded overhangs in wild-type cells by regulating telomere protein homeostasis. However, the absence of yKu70 may provide other proteins such as those involved in homologous recombination, Sgs1, or Pif1 additional access to G-rich, single-stranded DNA and may determine telomere size, cell cycle checkpoint activation, and, ultimately, temperature sensitivity.     

Dynamics of homologous chromosome pairing during meiotic prophase in fission yeast

Pairing of homologous chromosomes is important for homologous recombination and correct chromosome segregation during meiosis. It has been proposed that telomere clustering, nuclear oscillation, and recombination during meiotic prophase facilitate homologous chromosome pairing in fission yeast. Here we examined the contributions of these chromosomal events to homologous chromosome pairing, by directly observing the dynamics of chromosomal loci in living cells of fission yeast. Homologous loci exhibited a dynamic process of association and dissociation during the time course of meiotic prophase. Lack of nuclear oscillation reduced association frequency for both centromeric and arm regions of the chromosome. Lack of telomere clustering or recombination reduced association frequency at arm regions, but not significantly at centromeric regions. Our results indicate that homologous chromosomes are spatially aligned by oscillation of telomere-bundled chromosomes and physically linked by recombination at chromosome arm regions; this recombination is not required for association of homologous centromeres.     

Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication

Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.     

Homologous recombination generates T-loop-sized deletions at human telomeres

The t-loop structure of mammalian telomeres is thought to repress nonhomologous end joining (NHEJ) at natural chromosome ends. Telomere NHEJ occurs upon loss of TRF2, a telomeric protein implicated in t-loop formation. Here we describe a mutant allele of TRF2, TRF2DeltaB, that suppressed NHEJ but induced catastrophic deletions of telomeric DNA. The deletion events were stochastic and occurred rapidly, generating dramatically shortened telomeres that were accompanied by a DNA damage response and induction of senescence. TRF2DeltaB-induced deletions depended on XRCC3, a protein implicated in Holliday junction resolution, and created t-loop-sized telomeric circles. These telomeric circles were also detected in unperturbed cells and suggested that t-loop deletion by homologous recombination (HR) might contribute to telomere attrition. Human ALT cells had abundant telomeric circles, pointing to frequent t-loop HR events that could promote rolling circle replication of telomeres in the absence of telomerase. These findings show that t-loop deletion by HR influences the integrity and dynamics of mammalian telomeres.     

Developmentally regulated initiation of DNA synthesis by telomerase: evidence for factor-assisted de novo telomere formation.

Telomerase serves a dual role at telomeres, maintaining tracts of telomere repeats and forming telomeres de novo on broken chromosomes in a process called chromosome healing. In ciliates, both mechanisms are readily observed. Vegetatively growing cells maintain pre-existing telomeres, while cells undergoing macronuclear development fragment their chromosomes and form telomeres de novo. Here we provide the first evidence for developmentally regulated initiation of DNA synthesis by telomerase. In vitro assays were conducted with telomerase from vegetative and developing Euplotes macronuclei using chimeric primers that contained non-telomeric 3' ends and an upstream stretch of telomeric DNA. In developing macronuclei, chimeric primers had two fates: nucleotides were either polymerized directly onto the 3' terminus or residues were removed from the 3' end by endonucleolytic cleavage before polymerization began. In contrast, telomerase from vegetative macronuclei used only the cleavage pathway. Telomere repeat addition onto non-telomeric 3' ends was lost when developing macronuclei were lysed and the contents purified on glycerol gradients. However, when fractions from the glycerol gradient were added back to partially purified telomerase, telomere synthesis was restored. The data indicate that a dissociable chromosome healing factor (CHF) collaborates with telomerase to initiate developmentally programmed de novo telomere formation.     

The fission yeast Taz1 protein protects chromosomes from Ku-dependent end-to-end fusions

A paramount role of telomeres is to prevent chromosome fusions. The fission yeast Taz1 protein regulates diverse telomere functions but is not essential for growth under stress-free conditions. Strikingly, however, taz1(-) cells exhibit lethal telomere fusions when subjected to nitrogen starvation, a treatment that induces an uncommitted G1 state. These fusions are formed by Ku-dependent nonhomologous end joining. Fusions also occur during normal growth in taz1(-) cells that lack rad22(+), a gene involved in homologous recombination. Our data suggest a model whereby taz1(-) telomeres are exposed to the prevailing mode of DNA repair, which is dictated by the cell cycle. Thus, Taz1 caps chromosome ends and provides the telomerespecific interaction that prevents Ku from treating telomeres as double-strand breaks.     

De novo synthesis of telomere sequences at the healed breakpoints of wheat deletion chromosomes

When chromosomes are broken, the breakpoints become highly unstable and acquire the ability to fuse with other broken ends. The breakpoints are, however, eventually stabilized, and, therefore, the broken chromosomes are transmitted to the daughter cells without further morphological change. This phenomenon, known as “healing of breakpoints”, involves the addition of repetitive telomere sequences at the breakpoints by telomerase, the enzyme that normally synthesizes the telomere sequence at normal chromosome terminals. In many higher organisms, however, this property has not been well investigated. In this study, we examined the telomere sequences in wheat deletion lines with breakpoints on chromosome 1B. Lines that had breakpoints around the nucleolar organizer region were first selected on the basis of cytological observations, and the precise breakpoints were determined by mapping a fragment of rDNA and RFLP markers. In three lines – in addition to one previously reported – the DNA fragments encompassing the breakpoints were amplified by PCR using primers located in the rDNA and in telomere sequences. The DNA sequences provide insight into the properties of the telomerase activity at the breakpoints. The telomere sequences initiated from 2- to 4-nucleotide motifs in the original ribosomal DNA sequence which are also found in the repeat unit characteristic of telomere sequences. No specific sequences or structures were observed at or around the breakpoints. At all of the four breakpoints investigated, the newly synthesized telomere sequences contained considerable numbers of atypical telomere sequence units, particularly TTAGGG, which is the common unit of mammalian telomere sequences. Based on these results, we discuss the ability of plant telomerase to initiate the de novo synthesis of telomere sequences at internal breakpoints. Received: 15 June 1999 / Accepted: 6 August 1999     

Developmentally programmed healing of chromosomes by telomerase in Tetrahymena

Healing of a broken chromosome and in eukaryotes involves acquisition of a telomere. During macronuclear development in ciliated protozoans, germline chromosomes are fragmented into linear subchromosomes, whose ends are healed by de novo addition of telomeres. We showed previously that the ribonucleoprotein enzyme telomerase elongates preexisting telomeres by synthesizing one telomeric DNA strand, using a template sequence in the RNA moiety of the enzyme. By marking telomerase with a mutation in the telomerase RNA template, which causes synthesis of novel telomeric sequences, we now show that in the ciliate Tetrahymena, telomerase directly adds telomeric DNA onto nontelomeric sequences during developmentally controlled chromosome healing. Unexpectedly, one telomerase RNA template mutation converted telomerase from an enzyme that normally synthesizes precisely templated sequences to a less precise polymerase that sometimes synthesizes irregular telomeric repeats in vivo.     

The Pif1 Helicase,a Negative Regulator of Telomerase,Acts Preferentially at Long Telomeres

Telomerase, the enzyme that maintains telomeres, preferentially lengthens short telomeres. The S. cerevisiae Pif1 DNA helicase inhibits both telomerase-mediated telomere lengthening and de novo telomere addition at double strand breaks (DSB). Here, we report that the association of the telomerase subunits Est2 and Est1 at a DSB was increased in the absence of Pif1, as it is at telomeres, suggesting that Pif1 suppresses de novo telomere addition by removing telomerase from the break. To determine how the absence of Pif1 results in telomere lengthening, we used the single telomere extension assay (STEX), which monitors lengthening of individual telomeres in a single cell cycle. In the absence of Pif1, telomerase added significantly more telomeric DNA, an average of 72 nucleotides per telomere compared to the 45 nucleotides in wild type cells, and the fraction of telomeres lengthened increased almost four-fold. Using an inducible short telomere assay, Est2 and Est1 no longer bound preferentially to a short telomere in pif1 mutant cells while binding of Yku80, a telomere structural protein, was unaffected by the status of the PIF1 locus. Two experiments demonstrate that Pif1 binding is affected by telomere length: Pif1 (but not Yku80) -associated telomeres were 70 bps longer than bulk telomeres, and in the inducible short telomere assay, Pif1 bound better to wild type length telomeres than to short telomeres. Thus, preferential lengthening of short yeast telomeres is achieved in part by targeting the negative regulator Pif1 to long telomeres.     

Telomerase and ATM/Tel1p protect telomeres from nonhomologous end joining

Telomeres protect chromosome ends from fusing to double-stranded breaks (DSBs). Using a quantitative real-time PCR assay, we show that nonhomologous end joining between a telomere and an inducible DSB was undetectable in wild-type cells, but occurred within a few hours of DSB induction in approximately 1/2000 genomes in telomerase-deficient cells and in >1/1000 genomes in telomerase-deficient cells also lacking the ATM homolog Tel1p. The fused telomeres contained very little telomeric DNA, suggesting that catastrophic telomere shortening preceded fusion. Lengthening of telomeres did not prevent such catastrophic telomere shortening and fusion events. Telomere-DSB fusion also occurred in cells containing a catalytically inactive telomerase and in tel1 mec1 cells where telomerase cannot elongate telomeres. Thus, telomerase and Tel1p function in telomere protection as well as in telomere elongation.     

Saccharomyces cerevisiae as a model system to define the chromosomal instability phenotype

Translocations, deletions, and chromosome fusions are frequent events seen in cancers with genome instability. Here we analyzed 358 genome rearrangements generated in Saccharomyces cerevisiae selected by the loss of the nonessential terminal segment of chromosome V. The rearrangements appeared to be generated by both nonhomologous end joining and homologous recombination and targeted all chromosomes. Fifteen percent of the rearrangements occurred independently more than once. High levels of specific classes of rearrangements were isolated from strains with specific mutations: translocations to Ty elements were increased in telomerase-defective mutants, potential dicentric translocations and dicentric isochromosomes were associated with cell cycle checkpoint defects, chromosome fusions were frequent in strains with both telomerase and cell cycle checkpoint defects, and translocations to homolog genes were seen in strains with defects allowing homoeologous recombination. An analysis of human cancer-associated rearrangements revealed parallels to the effects that strain genotypes have on classes of rearrangement in S. cerevisiae.