The Caenorhabditis elegans Rad17 homolog HPR-17 is required for telomere replication

Subunits of the Rad9/Rad1/Hus1 (9-1-1) proliferating cell nuclear antigen (PNCA)-like sliding clamp are required for DNA damage responses and telomerase-mediated telomere replication in the nematode Caenorhabditis elegans. PCNA sliding clamps are loaded onto DNA by a replication factor C (RFC) clamp loader. The C. elegans Rad17 RFC clamp loader homolog, hpr-17, functions in the same pathway as the 9-1-1 complex with regard to both the DNA damage response and telomerase-mediated telomere elongation. Thus, hpr-17 defines an RFC-like complex that facilitates telomerase activity in vivo in C. elegans.     

Activation of Mrc1, a mediator of the replication checkpoint, by telomere erosion

Background information. In budding yeast, the loss of either telomere sequences (in telomerase‐negative cells) or telomere capping (in mutants of two telomere end‐protection proteins, Cdc13 and Yku) lead, by distinct pathways, to telomeric senescence. After DNA damage, activation of Rad53, which together with Chk1 represents a protein kinase central to all checkpoint pathways, normally requires Rad9, a checkpoint adaptor. Results. We report that in telomerase‐negative (tlc1Δ) cells, activation of Rad53, although diminished, could still take place in the absence of Rad9. In contrast, Rad9 was essential for Rad53 activation in cells that entered senescence in the presence of functional telomerase, namely in senescent cells bearing mutations in telomere end‐protection proteins (cdc13‐1 yku70Δ). In telomerase‐negative cells deleted for RAD9, Mrc1, another checkpoint adaptor previously implicated in the DNA replication checkpoint, mediated Rad53 activation. Rad9 and Rad53, as well as other DNA damage checkpoint proteins (Mec1, Mec3, Chk1 and Dun1), were required for complete DNA‐damage‐induced cell‐cycle arrest after loss of telomerase function. However, unexpectedly, given the formation of an active Rad53–Mrc1 complex in tlc1Δ rad9Δ cells, Mrc1 did not mediate the cell‐cycle arrest elicited by telomerase loss. Finally, we report that Rad9, Mrc1, Dun1 and Chk1 are activated by phosphorylation after telomerase inactivation. Conclusions. These results indicate that loss of telomere capping and loss of telomere sequences, both of which provoke telomeric senescence, are perceived as two distinct types of damages. In contrast with the Rad53–Rad9‐mediated cell‐cycle arrest that functions in a similar way in both types of telomeric senescence, activation of Rad53–Mrc1 might represent a specific response to telomerase inactivation and/or telomere shortening, the functional significance of which has yet to be uncovered.     

The Rad51 pathway of telomerase-independent maintenance of telomeres can amplify TG1-3 sequences in yku and cdc13 mutants of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, Cdc13, Yku, and telomerase define three parallel pathways for telomere end protection that prevent chromosome instability and death by senescence. We report here that cdc13-1 yku70delta mutants generated telomere deprotection-resistant cells that, in contrast with telomerase-negative senescent cells, did not display classical crisis events. cdc13-1 yku70delta cells survived telomere deprotection by exclusively amplifying TG(1-3) repeats (type II recombination). In a background lacking telomerase (tlc1delta), this process predominated over type I recombination (amplification of subtelomeric Y' sequences). Strikingly, inactivation of the Rad50/Rad59 pathway (which is normally required for type II recombination) in cdc13-1 yku70delta or yku70delta tlc1delta mutants, but also in cdc13-1 YKU70(+) tlc1delta mutants, still permitted type II recombination, but this process was now entirely dependent on the Rad51 pathway. In addition, delayed senescence was observed in cdc13-1 yku70delta rad51delta and cdc13-1 tlc1delta rad51delta cells. These results demonstrate that in wild-type cells, masking by Cdc13 and Yku prevents the Rad51 pathway from amplifying telomeric TG(1-3) sequences. They also suggest that Rad51 is more efficient than Rad50 in amplifying the sequences left uncovered by the absence of Cdc13 or Yku70.     

Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres

Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance. Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization. Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway. However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes. Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1Delta mutant, suggesting that these pathways may contribute to telomere protection. Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres. Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres. Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres.     

New function of CDC13 in positive telomere length regulation

Two roles for the Saccharomyces cerevisiae Cdc13 protein at the telomere have previously been characterized: it recruits telomerase to the telomere and protects chromosome ends from degradation. In a synthetic lethality screen with YKU70, the 70-kDa subunit of the telomere-associated Yku heterodimer, we identified a new mutation in CDC13, cdc13-4, that points toward an additional regulatory function of CDC13. Although CDC13 is an essential telomerase component in vivo, no replicative senescence can be observed in cdc13-4 cells. Telomeres of cdc13-4 mutants shorten for about 150 generations until they reach a stable level. Thus, in cdc13-4 mutants, telomerase seems to be inhibited at normal telomere length but fully active at short telomeres. Furthermore, chromosome end structure remains protected in cdc13-4 mutants. Progressive telomere shortening to a steady-state level has also been described for mutants of the positive telomere length regulator TEL1. Strikingly, cdc13-4/tel1Delta double mutants display shorter telomeres than either single mutant after 125 generations and a significant amplification of Y' elements after 225 generations. Therefore CDC13, TEL1, and the Yku heterodimer seem to represent distinct pathways in telomere length maintenance. Whereas several CDC13 mutants have been reported to display elongated telomeres indicating that Cdc13p functions in negative telomere length control, we report a new mutation leading to shortened and eventually stable telomeres. Therefore we discuss a key role of CDC13 not only in telomerase recruitment but also in regulating telomerase access, which might be modulated by protein-protein interactions acting as inhibitors or activators of telomerase activity.     

Genome integrity is regulated by the Caenorhabditis elegans Rad51D homolog rfs-1

Multiple mechanisms ensure genome maintenance through DNA damage repair, suppression of transposition, and telomere length regulation. The mortal germline (Mrt) mutants in Caenorhabditis elegans are defective in maintaining genome integrity, resulting in a progressive loss of fertility over many generations. Here I show that the high incidence of males (him)-15 locus, defined by the deficiency eDf25, is allelic to rfs-1, the sole rad-51 paralog group member in C. elegans. The rfs-1/eDf25 mutant displays a Mrt phenotype and mutant animals manifest features of chromosome fusions prior to the onset of sterility. Unlike other Mrt genes, rfs-1 manifests fluctuations in telomere lengths and functions independently of telomerase. These data suggest that rfs-1 is a novel regulator of genome stability.     

Mutant telomere sequences lead to impaired chromosome separation and a unique checkpoint response

Mutation of the template region in the RNA component of telomerase can cause incorporation of mutant DNA sequences at telomeres. We made all 63 mutant sequence combinations at template positions 474-476 of the yeast telomerase RNA, TLC1. Mutants contained faithfully incorporated template mutations, as well as misincorporated sequences in telomeres, a phenotype not previously reported for Saccharomyces cerevisiae telomerase template mutants. Although growth rates and telomere profiles varied widely among the tlc1 mutants, chromosome separation and segregation were always aberrant. The mutants showed defects in sister chromatid separation at centromeres as well as telomeres, suggesting activation of a cell cycle checkpoint. Deletion of the DNA damage response genes DDC1, MEC3, or DDC2/SML1 failed to restore chromosome separation in the tlc1 template mutants. These results suggest that mutant telomere sequences elicit a checkpoint that is genetically distinct from those activated by deletion of telomerase or DNA damage.     

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.     

Fission yeast Taz1 and RPA are synergistically required to prevent rapid telomere loss

The telomere complex must allow nucleases and helicases to process chromosome ends to make them substrates for telomerase, while preventing these same activities from disrupting chromosome end-protection. Replication protein A (RPA) binds to single-stranded DNA and is required for DNA replication, recombination, repair, and telomere maintenance. In fission yeast, the telomere binding protein Taz1 protects telomeres and negatively regulates telomerase. Here, we show that taz1-d rad11-D223Y double mutants lose their telomeric DNA, indicating that RPA (Rad11) and Taz1 are synergistically required to prevent telomere loss. Telomere loss in the taz1-d rad11-D223Y double mutants was suppressed by additional mutation of the helicase domain in a RecQ helicase (Rqh1), or by overexpression of Pot1, a single-strand telomere binding protein that is essential for protection of chromosome ends. From our results, we propose that in the absence of Taz1 and functional RPA, Pot1 cannot function properly and the helicase activity of Rqh1 promotes telomere loss. Our results suggest that controlling the activity of Rqh1 at telomeres is critical for the prevention of genomic instability.     

Sgs1 RecQ helicase inhibits survival of Saccharomyces cerevisiae cells lacking telomerase and homologous recombination

In yeast telomerase mutants, the Sgs1 RecQ helicase slows the rate of senescence and also facilitates the appearance of certain types of survivors of critical telomere shortening via mechanisms dependent on Rad52-dependent homologous recombination (HR). Here we describe a third function for Sgs1 in telomerase-deficient cells, inhibition of survivors that grow independent of Rad52. Unlike tlc1 rad52 double mutants, which do not form survivors of telomere dysfunction, tlc1 rad52 sgs1 triple mutants readily generated survivors. After emerging from growth crisis, the triple mutants progressively lost telomeric and subtelomeric sequences, yet grew for more than 1 year. Analysis of cloned chromosome termini and of copy number changes of loci genome-wide using tiling arrays revealed terminal deletions extending up to 57 kb, as well as changes in Ty retrotransposon copy numbers. Amplification of the remaining terminal sequences generated large palindromes at some chromosome termini. Sgs1 helicase activity but not checkpoint function was essential for inhibiting the appearance of the survivors, and the continued absence of Sgs1 was required for the growth of the established survivors. Thus, in addition to facilitating the maintenance of telomere repeat sequences via HR-dependent mechanisms, a RecQ helicase can prevent the adoption of HR-independent mechanisms that stabilize chromosome termini without the use of natural telomere sequences. This provides a novel mechanism by which RecQ helicases may help maintain genome integrity and thus prevent age-related diseases and cancer.     

The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability

The Mre11/Rad50/Nbs (MRN) complex and the two protein kinases ATM and ATR play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. It has been previously shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. Here, we have analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations.     

RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.     

Exonuclease activity is required for sequence addition and Cdc13p loading at a de novo telomere.

The Saccharomyces cerevisiae Mre11p/Rad50p/Xrs2p (MRX) complex is evolutionarily conserved and functions in DNA repair and at telomeres [1-3]. In vivo, MRX is required for a 5' --> 3' exonuclease activity that mediates DNA recombination at double-strand breaks (DSBs). Paradoxically, abolition of this exonuclease activity in MRX mutants results in shortened telomeric DNA tracts. To further explore the role of MRX at telomeres, we analyzed MRX mutants in a de novo telomere addition assay in yeast cells [4]. We found that the MRX genes were absolutely required for telomerase-mediated addition in this assay. Furthermore, we found that Cdc13p, a single-stranded telomeric DNA binding protein essential for telomere DNA synthesis and protection [5], was unable to bind to the de novo telomeric DNA substrate in cells lacking Rad50p. Based on the results from this model system, we propose that the MRX complex helps to prepare telomeric DNA for the loading of Cdc13p, which then protects the chromosome from further degradation and recruits telomerase and other DNA replication components to synthesize telomeric DNA.     

Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13

In Saccharomyces cerevisiae, Cdc13 has been proposed to mediate telomerase recruitment at telomere ends. Stn1, which associates with Cdc13 by the two-hybrid interaction, has been implicated in telomere maintenance. Ten1, a previously uncharacterized protein, was found to associate physically with both Stn1 and Cdc13. A binding defect between Stn1-13 and Ten1 was responsible for the long telomere phenotype of stn1-13 mutant cells. Moreover, rescue of the cdc13-1 mutation by STN1 was much improved when TEN1 was simultaneously overexpressed. Several ten1 mutations were found to confer telomerase-dependent telomere lengthening. Other, temperature-sensitive, mutants of TEN1 arrested at G(2)/M via activation of the Rad9-dependent DNA damage checkpoint. These ten1 mutant cells were found to accumulate single-stranded DNA in telomeric regions of the chromosomes. We propose that Ten1 is required to regulate telomere length, as well as to prevent lethal damage to telomeric DNA.     

Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae.

To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length.