Telomere Maintenance In Yeast Research Articles

Set1 regulates telomere function via H3K4 methylation-dependent and -independent pathways and calibrates the abundance of telomere maintenance factors.

Set1 is an H3K4 methyltransferase that comprises the catalytic subunit of the COMPASS complex and has been implicated in transcription, DNA repair, cell cycle control, and numerous other genomic functions. Set1 also promotes proper telomere maintenance, as cells lacking Set1 have short telomeres and disrupted subtelomeric gene repression; however, the precise role for Set1 in these processes has not been fully defined. In this study, we have tested mutants of Set1 and the COMPASS complex that differentially alter H3K4 methylation status, and we have attempted to separate catalytic and noncatalytic functions of Set1. Our data reveal that Set1-dependent subtelomeric gene repression relies on its catalytic activity toward H3K4, whereas telomere length is regulated by Set1 catalytic activity but likely independent of the H3K4 substrate. Furthermore, we uncover a role for Set1 in calibrating the abundance of critical telomere maintenance proteins, including components of the telomerase holoenzyme and members of the telomere capping CST (Cdc13-Stn1-Ten1) complex, through both transcriptional and posttranscriptional pathways. Altogether, our data provide new insights into the H3K4 methylation-dependent and -independent roles for Set1 in telomere maintenance in yeast and shed light on possible roles for Set1-related methyltransferases in other systems.

ORCA/LRWD1 Regulates Homologous Recombination at ALT-Telomeres by Modulating Heterochromatin Organization.

SummaryTelomeres are maintained by telomerase or in a subset of cancer cells by a homologous recombination (HR)-based mechanism, Alternative Lengthening of Telomeres (ALT). The mechanisms regulating telomere-homeostasis in ALT cells remain unclear. We report that a replication initiator protein, Origin Recognition Complex-Associated (ORCA/LRWD1), by localizing at the ALT-telomeres, modulates HR activity. ORCA's localization to the ALT-telomeres is facilitated by its interaction to SUMOylated shelterin components. The loss of ORCA in ALT-positive cells elevates the levels of two mediators of HR, RPA and RAD51, and consistent with this, we observe increased ALT-associated promyelocytic leukemia body formation and telomere sister chromatid exchange. ORCA binds to RPA and modulates the association of RPA to telomeres. Finally, the loss of ORCA causes global chromatin decondensation, including at the telomeres. Our results demonstrate that ORCA acts as an inhibitor of HR by modulating RPA binding to ssDNA and inducing chromatin compaction.

WGS-based telomere length analysis in Dutch family trios implicates stronger maternal inheritance and a role for RRM1 gene

Telomere length (TL) regulation is an important factor in ageing, reproduction and cancer development. Genetic, hereditary and environmental factors regulating TL are currently widely investigated, however, their relative contribution to TL variability is still understudied. We have used whole genome sequencing data of 250 family trios from the Genome of the Netherlands project to perform computational measurement of TL and a series of regression and genome-wide association analyses to reveal TL inheritance patterns and associated genetic factors. Our results confirm that TL is a largely heritable trait, primarily with mother’s, and, to a lesser extent, with father’s TL having the strongest influence on the offspring. In this cohort, mother’s, but not father’s age at conception was positively linked to offspring TL. Age-related TL attrition of 40 bp/year had relatively small influence on TL variability. Finally, we have identified TL-associated variations in ribonuclease reductase catalytic subunit M1 (RRM1 gene), which is known to regulate telomere maintenance in yeast. We also highlight the importance of multivariate approach and the limitations of existing tools for the analysis of TL as a polygenic heritable quantitative trait.

Single-stranded telomere-binding protein employs a dual rheostat for binding affinity and specificity that drives function

ssDNA, which is involved in numerous aspects of chromosome biology, is managed by a suite of proteins with tailored activities. The majority of these proteins bind ssDNA indiscriminately, exhibiting little apparent sequence preference. However, there are several notable exceptions, including the Saccharomyces cerevisiae Cdc13 protein, which is vital for yeast telomere maintenance. Cdc13 is one of the tightest known binders of ssDNA and is specific for G-rich telomeric sequences. To investigate how these two different biochemical features, affinity and specificity, contribute to function, we created an unbiased panel of alanine mutations across the Cdc13 DNA-binding interface, including several aromatic amino acids that play critical roles in binding activity. A subset of mutant proteins exhibited significant loss in affinity in vitro that, as expected, conferred a profound loss of viability in vivo. Unexpectedly, a second category of mutant proteins displayed an increase in specificity, manifested as an inability to accommodate changes in ssDNA sequence. Yeast strains with specificity-enhanced mutations displayed a gradient of viability in vivo that paralleled the loss in sequence tolerance in vitro, arguing that binding specificity can be fine-tuned to ensure optimal function. We propose that DNA binding by Cdc13 employs a highly cooperative interface whereby sequence diversity is accommodated through plastic binding modes. This suggests that sequence specificity is not a binary choice but rather is a continuum. Even in proteins that are thought to be specific nucleic acid binders, sequence tolerance through the utilization of multiple binding modes may be a broader phenomenon than previously appreciated.

Rnr1's role in telomere elongation cannot be replaced by Rnr3: a role beyond dNTPs?

Telomeres, the nucleoprotein complexes at the end of eukaryotic chromosomes, protect them from degradation and ensure the replicative capacity of cells. In most human tumors and in budding yeast, telomere length is maintained by the activity of telomerase, an enzyme that adds dNTPs according to an internal RNA template. The dNTPs are generated with the help of the ribonucleotide reductase (RNR) complex. We have recently generated strains lacking the large subunit of RNR, Rnr1, which were kept viable by the expression of RNR complexes containing the Rnr1 homolog, Rnr3. Interestingly, we found that these Rnr1-deficient strains have short telomeres that are stably maintained, but cannot become efficiently elongated by telomerase. Thus, a basic maintenance of short telomeres is possible under conditions, where Rnr1 activity is absent, but a sustained elongation of short telomeres fully depends on Rnr1 activity. We show that Rnr3 cannot compensate for this telomeric function of Rnr1 even when overall cellular dNTP values are restored. This suggests that Rnr1 plays a role in telomere elongation beyond increasing cellular dNTP levels. Furthermore, our data indicate that telomerase may act in two different modes, one that is capable of coping with the "end-replication problem" and is functional even in the absence of Rnr1 and another required for the sustained elongation of short telomeres, which fully depends on the presence of Rnr1. Supply of dNTPs for telomere elongation is provided by the Mec1ATR checkpoint, both during regular DNA replication and upon replication fork stalling. We discuss the implications of these results on telomere maintenance in yeast and cancer cells.

Yeast telomere maintenance is globally controlled by programmed ribosomal frameshifting and the nonsense-mediated mRNA decay pathway

We have previously shown that ~10% of all eukaryotic mRNAs contain potential programmed -1 ribosomal frameshifting (-1 PRF) signals and that some function as mRNA destabilizing elements through the Nonsense-Mediated mRNA Decay (NMD) pathway by directing translating ribosomes to premature termination codons. Here, the connection between -1 PRF, NMD and telomere end maintenance are explored. Functional -1 PRF signals were identified in the mRNAs encoding two components of yeast telomerase, EST1 and EST2, and in mRNAs encoding proteins involved in recruiting telomerase to chromosome ends, STN1 and CDC13. All of these elements responded to mutants and drugs previously known to stimulate or inhibit -1 PRF, further supporting the hypothesis that they promote -1 PRF through the canonical mechanism. All affected the steady-state abundance of a reporter mRNA and the wide range of -1 PRF efficiencies promoted by these elements enabled the determination of an inverse logarithmic relationship between -1 PRF efficiency and mRNA accumulation. Steady-state abundances of the endogenous EST1, EST2, STN1 and CDC13 mRNAs were similarly inversely proportional to changes in -1 PRF efficiency promoted by mutants and drugs, supporting the hypothesis that expression of these genes is post-transcriptionally controlled by -1 PRF under native conditions. Overexpression of EST2 by ablation of -1 PRF signals or inhibition of NMD promoted formation of shorter telomeres and accumulation of large budded cells at the G2/M boundary. A model is presented describing how limitation and maintenance of correct stoichiometries of telomerase components by -1 PRF is used to maintain yeast telomere length.

Natural Polymorphism in BUL2 Links Cellular Amino Acid Availability with Chronological Aging and Telomere Maintenance in Yeast

Aging and longevity are considered to be highly complex genetic traits. In order to gain insight into aging as a polygenic trait, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard strain RM11 and a laboratory strain S288c, to identify quantitative trait loci that control chronological lifespan. Among the major loci that regulate chronological lifespan in this cross, one genetic linkage was found to be congruent with a previously mapped locus that controls telomere length variation. We found that a single nucleotide polymorphism in BUL2, encoding a component of an ubiquitin ligase complex involved in trafficking of amino acid permeases, controls chronological lifespan and telomere length as well as amino acid uptake. Cellular amino acid availability changes conferred by the BUL2 polymorphism alter telomere length by modulating activity of a transcription factor Gln3. Among the GLN3 transcriptional targets relevant to this phenotype, we identified Wtm1, whose upregulation promotes nuclear retention of ribonucleotide reductase (RNR) components and inhibits the assembly of the RNR enzyme complex during S-phase. Inhibition of RNR is one of the mechanisms by which Gln3 modulates telomere length. Identification of a polymorphism in BUL2 in this outbred yeast population revealed a link among cellular amino acid availability, chronological lifespan, and telomere length control.

Abrupt telomere losses and reduced end-resection can explain accelerated senescence of Smc5/6 mutants lacking telomerase

The highly conserved Structural Maintenance of Chromosome (SMC) proteins are crucial for the formation of three essential complexes involved in high fidelity chromosome transmission during cell division. Recently, the Smc5/6 complex has been reported to be important for telomere maintenance in yeast and also in cancerous human ALT cells, where it could function in a homologous recombination-based (HR) telomere maintenance pathway. Here, we investigate the possible roles of the budding yeast Smc5/6 complex in maintaining appropriate chromosome end-structures allowing cell survival in absence of telomerase. The results show that cells harbouring mutant alleles of genes encoding Smc5/6-complex proteins rapidly stop growing after telomerase loss. Furthermore, this telomerase-induced growth arrest is much more pronounced as compared to cultures with a functional Smc5/6-complex. Bulk telomere sequence loss is not increased in the mutant cells and the evidence suggests that Smc5/6 slows senescence through a partially HR-independent pathway. We propose that in yeast, the Smc5/6-complex is required for efficient and timely termination of DNA replication and repair at telomeres to avoid stochastic telomere loss during cell division. Consistent with this hypothesis, sequencing of telomeres from telomerase-positive smc5/6 mutant cells revealed a higher frequency of telomere breakage events. Finally, the results also show that on dysfunctional telomeres, the generation of 3′-single stranded DNA is impaired, suggesting that the complex may also participate in the formation of single-stranded overhangs which are thought to be the substrates for telomere repeat replenishment in the absence of telomerase.

Roles of the checkpoint sensor clamp Rad9-Rad1-Hus1 (911)-complex and the clamp loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe telomere maintenance

While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.

A role for Brca1 in chromosome end maintenance

The role of BRCA1 in breast and ovarian tumor suppression has been primarily ascribed to the maintenance of genome integrity. BRCA1 interacts with components of the non-homologous end-joining pathway previously shown to play a role in telomere maintenance in yeast. Here, we provide evidence that links Brca1 with telomere integrity. Brca1(-/-) T-cells display telomere dysfunction in both loss of telomere repeats as well as defective telomere capping. Loss of Brca1 synergizes with p53 deficiency in the onset and frequency of tumorigenesis. Karyotyping of tBrca1(-/-)p53(-/-) thymic lymphomas revealed the presence of telomere dysfunction accompanied by clonal chromosomal translocations. The telomere dysfunction phenotype in Brca1-deficient cells suggests that loss of telomere integrity might contribute to chromosome end dysfunction and permit the formation of potentially oncogenic translocations.

The Mre11p/Rad50p/Xrs2p complex and the Tel1p function in a single pathway for telomere maintenance in yeast.

The Mre11p/Rad50p/Xrs2p complex is involved in the repair of double-strand DNA breaks, nonhomologous end joining, and telomere length regulation. TEL1 is primarily involved in telomere length regulation. By an epistasis analysis, we conclude that Tel1p and the Mre11p/Rad50p/Xrs2p complex function in a single pathway of telomere length regulation.

The Mre11p/Rad50p/Xrs2p complex and the Tel1p function in a single pathway for telomere maintenance in yeast.

The Mre11p/Rad50p/Xrs2p complex and the Tel1p function in a single pathway for telomere maintenance in yeast.

Ku is associated with the telomere in mammals.

Telomeres are specialized DNA/protein complexes that comprise the ends of eukaryotic chromosomes. The highly expressed Ku heterodimer, composed of 70 and 80 K(d) subunits (Ku70 and Ku80), is the high-affinity DNA binding component of the DNA-dependent protein kinase. Ku is critical for nonhomologous DNA double-stranded break repair and site-specific recombination of V(D)J gene segments. Ku also plays an important role in telomere maintenance in yeast. Herein, we report, using an in vivo crosslinking method, that human and hamster telomeric DNAs specifically coimmunoprecipitate with human Ku80 after crosslinking. Localization of Ku to the telomere does not depend on the DNA-dependent protein kinase catalytic component. These findings suggest a direct link between Ku and the telomere in mammalian cells.

Y′-Help1, a DNA Helicase Encoded by the Yeast Subtelomeric Y′ Element, Is Induced in Survivors Defective for Telomerase

The yeast Y' element is a highly polymorphic repetitive sequence present in the subtelomeric regions of many yeast telomeres. The Y' element is classed as either Y'-L or Y'-S, depending on its length. It has been reported that survivors arising from telomerase-deficient yeast mutants compensate for telomere loss by the amplification of Y' elements. The total Saccharomyces cerevisiae genome DNA data base was searched for Y' elements, and 11 Y'-Ls and eight Y'-Ss were identified. As reported previously, many of the sequences were found to contain long open reading frames which potentially encode helicase. We examined the expression of the Y' elements in telomerase-deficient Deltatlc1 survivors, in which the TLC1 gene encoding the yeast telomerase template RNA had been disrupted, and found that the Y' element is highly expressed in the survivors, but not in the wild-type cells. Moreover, we demonstrated that the survivors produce a Y'-encoded protein designated as Y'-Help1 (Y'-helicase protein 1), and that this protein possesses helicase activity. Therefore, we suggest that the Y' element has a novel and potentially important role in trans, in addition to the well characterized role in cis, in telomerase-independent telomere maintenance in yeast.

Telomere maintenance is dependent on activities required for end repair of double-strand breaks.

Telomeres are functionally distinct from ends generated by chromosome breakage, in that telomeres, unlike double-strand breaks, are insulated from recombination with other chromosomal termini [1]. We report that the Ku heterodimer and the Rad50/Mre11/Xrs2 complex, both of which are required for repair of double-strand breaks [2-5], have separate roles in normal telomere maintenance in yeast. Using epistasis analysis, we show that the Ku end-binding complex defined a third telomere-associated activity, required in parallel with telomerase [6] and Cdc13, a protein binding the single-strand portion of telomere DNA [7,8]. Furthermore, loss of Ku function altered the expression of telomere-located genes, indicative of a disruption of telomeric chromatin. These data suggest that the Ku complex and the Cdc13 protein function as terminus-binding factors, contributing distinct roles in chromosome end protection. In contrast, MRE11 and RAD50 were required for the telomerase-mediated pathway, rather than for telomeric end protection; we propose that this complex functions to prepare DNA ends for telomerase to replicate. These results suggest that as a part of normal telomere maintenance, telomeres are identified as double-strand breaks, with additional mechanisms required to prevent telomere recombination. Ku, Cdc13 and telomerase define three epistasis groups required in parallel for telomere maintenance.

Cdc13p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance.

The CDC13 gene has previously been implicated in the maintenance of telomere integrity in Saccharomyces cerevisiae. With the use of two classes of mutations, here it is shown that CDC13 has two discrete roles at the telomere. The cdc13-2est mutation perturbs a function required in vivo for telomerase regulation but not in vitro for enzyme activity, whereas cdc13-1ts defines a separate essential role at the telomere. In vitro, purified Cdc13p binds to single-strand yeast telomeric DNA. Therefore, Cdc13p is a telomere-binding protein required to protect the telomere and mediate access of telomerase to the chromosomal terminus.

How does the end begin?: Formation and maintenance of telomeres in ciliates and yeast

Current models of telomere formation and replication involve either telomerase, a novel ribonucleoprotein, or recombination between the ends of DNA molecules. However, present models will have to be modified to explain recent data on telomere formation in yeast. An understanding of the mechanisms of telomere maintenance in yeast may reveal how other organisms with heterogeneous telomeric repeats replicate their chromosomal termini.