Telomerase Dependence of Telomere Lengthening in Ku80 Mutant Arabidopsis

Telomerase Dependence of Telomere Lengthening in ku80 Mutant Arabidopsis

Maria Eugenia Gallego, Nicole Jalut, and Charles I. White1 Unité Mixte de Recherche 6547 du Centre National de la Recherche Scientifique, Université Blaise Pascal 24, 63177 Aubière, France

We have identified a ku80 mutant of Arabidopsis and show that telomerase is needed to generate the longer telomeres observed in this mutant. Telomeres are specialized nucleoprotein structures at the ends of chromosomes that permit cells to distinguish chromosome ends from double-strand breaks, thus preventing chromosome fusion events. Ku80 deficiency results in the lengthening of telomeres, a phenotype also seen in an Arabidopsis ku70 mutant. Furthermore, homogeneous populations of ku80 mutant cells show a steady increase in the length of telomere tracts, which reach an equilibrium length and then stabilize. In contrast to that in mammals, Ku80 deficiency in Arabidopsis cells does not cause end-to-end fusion of chromosomes. This telomere lengthening is dependent on the presence of telomerase, although it is not attributable to a significant increase in telomerase activity per se. These results demonstrate the essential role of the Ku80 protein as a negative regulator of telomerase function in plant cells.

INTRODUCTION two general mechanisms have been identified: homologous recombination and nonhomologous end joining (NHEJ). These Telomeres are specialized nucleoprotein structures at the ends mechanisms are present in species from yeast to mammals of chromosomes that protect them from fusion and degrada- and plants. However, yeast cells use mainly homologous re- tion. They consist of a repeated DNA sequence conforming to combination, whereas higher eukaryotes favor NHEJ (Paques the consensus Tx(A)Gy and an unknown number of proteins. and Haber, 1999; Khanna and Jackson, 2001; van Gent et al., Telomeric repeats are extended by telomerase, a reverse tran- 2001). scriptase with an RNA component that serves as a template for Several proteins have been identified in yeast and mammals the synthesis of de novo telomeric repeats. Telomere length is as essential for NHEJ. In yeast cells, the Ku heterodimer Ku70/ stable and species specific, suggesting mechanisms to regu- Ku80 and the Rad50-Mre11-Xrs2 complex play key roles in late telomerase and limit the addition of repeats. In mammals, NHEJ. These two complexes respectively recognize the DNA telomeres have been shown to end in a large duplex loop, DSB and prepare it to be ligated by the Ligase IV-Xrcc4 com- termed the telomeric T-loop (Griffith et al., 1999). Two proteins, plex. In mammalian cells, the DNA-PK complex, which consists TRF1 and TRF2, bind directly to the double-stranded telomeric of the Ku heterodimer associated with the DNA-PK catalytic DNA (Bilaud et al., 1997; van Steensel and de Lange, 1997; van subunit, senses DNA damage and is an integral component of Steensel et al., 1998; Smogorzewska et al., 2000). Two other the NHEJ system (Karran, 2000; Khanna and Jackson, 2001; proteins, TIN2 and hRAP1, specifically localize to mammalian Pierce et al., 2001). Mice mutated in any of these genes are ra- telomeres by binding to TRF1 and TRF2, respectively (Kim et diation sensitive, present growth retardation, and are deficient al., 1999; Li et al., 2000). Telomere structure and maintenance, in V(D)J recombination (Blunt et al., 1996; Nussenzweig et al., in general, have been the subject of a number of recent reviews 1996; Zhu et al., 1996; Gu et al., 1997; Ouyang et al., 1997; Gao (Evans and Lundblad, 2000; Blackburn, 2001; Shore, 2001; et al., 1998). Chan and Blackburn, 2002; de Lange, 2002; McKnight et al., In yeast cells, the Rad50-Mre11-Xrs2 complex and the Ku 2002). heterodimer also are involved in telomere metabolism. Yeast A fundamental question concerning telomere regulation is cells deficient in any of these genes present shorter but sta- the means by which cells distinguish between DNA double- ble telomeres (Boulton and Jackson, 1996, 1998; Porter et al., strand breaks (DSBs) and chromosome ends. DSBs are caused 1996; Kironmai and Muniyappa, 1997; Nugent et al., 1998). The by cellular processes such as replication as well as by DNA- role of the Ku80 protein in telomere regulation has been shown damaging agents such as ionizing radiation, and tight regula- recently in mammals. Telomere length is deregulated in Ku80 tory and repair systems have evolved to respond to these mutant mouse cells; in fact, contradictory results showing both breaks. DSB repair is performed by genetic recombination, and shortening and lengthening of telomeres have been reported. However, in both studies, a high frequency of end-to-end chromosome fusions was found, suggesting a role for the 1 To whom correspondence should be addressed. E-mail chwhite@ Ku80 protein in protecting telomeres from fusion (Bailey et univ-bpclermont.fr; fax 33-473-407-777. Article, publication date, and citation information can be found at al., 1999; Samper et al., 2000; d’Adda di Fagagna et al., www.plantcell.org/cgi/doi/10.1105/tpc.008623. 2001; Espejel et al., 2002).

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Plant homologs of proteins involved in NHEJ have been Agronomique Versailles Arabidopsis T-DNA insertion collection characterized. Arabidopsis Ku70 and Ku80 proteins form a het- using the flanking insertion site FLAG sequence database erodimer with DNA binding activity (Riha et al., 2002; Tamura et (Samson et al., 2002) and identified a putative mutant plant with al., 2002; West et al., 2002). Plants homozygous for T-DNA in- a T-DNA insertion in the KU80 gene. PCR analysis using oligo- sertions in these genes are hypersensitive to DNA-damaging nucleotides specific for the KU80 gene and the T-DNA showed agents (Bundock et al., 2002; Riha et al., 2002; West et al., that the T-DNA is inserted in exon 10 of the KU80 gene (Figure 2002). Arabidopsis homologs of Ligase IV and its interacting 1). Should any protein be produced by this mutant ku80 locus, protein Xrcc4 also have been characterized (West et al., 2000), it would lack the C-terminal 125 amino acids. The inserted T-DNA but no mutants have been analyzed to date. Arabidopsis mu- has a 564-bp inverted duplication of the T-DNA left border se- tants harboring a T-DNA insertion in the RAD50 gene are sterile quence downstream of the right border. The beginning of the and present a hyperrecombination phenotype (Gherbi et al., insertion left border has a three-nucleotide homology with the 2001). Furthermore, cultured cells derived from rad50 mutant KU80 sequence. The left border at the end of the T-DNA inser- plants are hypersensitive to methyl methanesulfonate (Gallego tion has a nine-nucleotide homology with the KU80 gene (Fig- et al., 2001). ure 1). The kanamycin resistance marker of the inserted T-DNA Telomere structure and regulation in plants has been re- in progeny of selfed heterozygotes segregates with a 3:1 ratio viewed recently (McKnight et al., 2002). An Arabidopsis telo- for resistance to sensitivity, as expected for a single-locus in- merase mutant presents progressive shortening of telomeres, sertion (282 kanamycin-resistant:102 kanamycin-sensitive plants; and late-generation mutants are defective in vegetative growth, 2 [1 df] 0.5). DNA gel blot analysis confirmed that the T-DNA with chromosome fusions occurring in 40% of the cells insertion is present at a single locus (data not shown). Plants (Fitzgerald et al., 1999; Riha et al., 2001). We have shown previ- homozygous for the T-DNA insertion develop normally and are ously that the Arabidopsis Rad50 protein is involved in plant fertile. telomere metabolism (Gallego and White, 2001). Mutant cells To investigate whether Ku80 plays a functional role in telo- present progressive shortening of telomeres associated with mere regulation in Arabidopsis, callus suspension cultures cell senescence. However, surviving mutant cells have longer were derived from two individual ku80 mutant plants and a telomeres than those of nonmutant RAD50/rad50 heterozygote wild-type parent as a control. The genotypes of the cultured control cells. During the preparation of this article, Riha et al. cells were verified by DNA gel blot analysis using a KU80 DNA (2002) and Bundock et al. (2002) reported the presence of long probe (data not shown). The telomere length of these cells was telomeres in Arabidopsis ku70 mutants, suggesting a role for measured by DNA gel blot analysis of MboI-digested genomic Ku70 in telomere metabolism. A telomere binding protein ho- DNA using the telomere repeat as a probe. Wild-type cells pre- mologous with the mammalian TRF1 protein has been identi- sented the expected telomeric smear, whereas cell lines de- fied in Arabidopsis, but no mutant has been studied (Chen et rived from homozygous ku80 plants showed much longer telo- al., 2001). meres (Figure 2A). Longer telomeres were observed after 11 Here, we present an analysis of the possible role of the Ku80 weeks of growth in ku80 cells compared with the wild type. Af- protein in telomere metabolism in plant cells. Arabidopsis plants homozygous for a T-DNA insertion in the KU80 gene develop and grow normally. However, they present progressive lengthening of telomeric repeats both in planta and in callus cultures in vitro. This telomere extension is dependent on the presence of telomerase, although it is not correlated with a significant increase in telomerase activity per se, as measured by the in vitro telomerase repeat amplification protocol (TRAP) assay. Thus, the Ku protein controls telomere length in Arabidopsis by directly or indirectly inhibiting the action of the telomerase at telomere ends.

RESULTS

The ku80 Mutant Presents Longer Telomeres in Figure 1. T-DNA Insertion into the Arabidopsis KU80 Gene. Plant Cells Boxes represent the exons in the KU80 gene. The position of the T-DNA Based on the Arabidopsis genome sequence, we used reverse insertion is indicated. At bottom, the junction sequences of the inserted transcriptase–mediated PCR to clone the cDNA of the Arabi- T-DNAs are shown. The three boxes show the three junctions: KU80– dopsis homolog of the human and yeast KU80 genes. The se- T-DNA, T-DNA–T-DNA, and T-DNA–KU80. The mutant ku80 genomic DNA sequence is aligned with the KU80 genomic sequence and the quence of our AtKU80 clone corresponds exactly to that sub- ends of the inserted T-DNA. The mutant sequence and the sequences mitted previously to GenBank by Y. Adachi, K. Oguchi, K. homologous with it (from which it derives) are shown in uppercase let- Tamura, and H. Takahashi. This cDNA gives a predicted protein ters. Orientations of the T-DNA sequence are indicated as right border– of 680 amino acids encoded by 12 exons. left border (rblb) and left border–right border (lbrb). LB, left border; RB, We then screened the Institut National de la Recherche right border.

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be the result of chromosome fusions. End-to-end chromosome fusions would result in the fused telomeric repeat sequences being internal to the chromosome. Thus, these fused telomeric repeats would become resistant to Bal31 exonuclease treatment. However, if the extended telomeres were generated by the addition of new repeats, they should be sensitive to Bal31 exonuclease attack. Genomic DNA from wild-type and ku80 mutant cells was treated with different concentrations of Bal31, digested with MboI, and analyzed by DNA gel blot hybridization with the telomere repeat probe. Bal31 digestion of genomic DNA from both ku80 mutant and wild-type cells showed progressive shortening of the telomeric repeats with increasing Bal31 concentration (Figure 3A). Thus, the longer telomeric sequences in the ku80 mutant cells were the result of lengthening of the telomeric repeats. Reprobing this blot with the chromosome II subtelomeric probe (see above) showed sharp, nonde- graded bands in the untreated lanes, confirming the integrity of the genomic DNA samples used (Figure 3B). These results are

Figure 2. Telomere Dynamics in Homogeneous Populations of ku80 Mutant Dividing Cells. DNA was prepared from wild-type (lanes ) and two independent ku80 (lanes a and b) cell cultures grown in liquid for the indicated number of weeks. MboI-digested DNA was examined by DNA gel blot analysis using the telomeric repeat probe (A). The DNA gel blot was washed off and reprobed with a subtelomeric region from the long arm of chromosome 2 (B), which specifically detects the telomeric MboI fragment of this chromosome arm.

ter 27 weeks, telomeres had reached their maximum length, and this length was stable for at least 34 weeks of growth. Fig- ure 2B shows the same DNA gel blot filter reprobed with a subtelomeric probe specific for the telomere of the long arm of chromosome 2 (Gallego and White, 2001). As reported previously for Arabidopsis (Gallego and White, 2001) and tobacco (Fajkus et al., 1998), telomere length was stable in wild-type cells, in contrast to the elongation reported upon culture in Me- landrium album (Riha et al., 1998) and barley (Kilian et al., 1995). Thus, the lack of a functional Ku80 protein in Arabidopsis re- sulted in a telomere elongation phenotype. The fact that ku80 Arabidopsis plants presented a telomere phenotype similar to that of ku70 plants, together with the known physical associa- tion of these two proteins, indicates that these two proteins work as a heterodimer for telomere length regulation (Bundock et al., 2002; Riha et al., 2002; Tamura et al., 2002; West et al., 2002). Figure 3. Arabidopsis ku80 Mutant Telomeres Are Sensitive to Bal31 Exonuclease. Genomic DNA from wild-type (WT) and ku80 mutant cells lines was Extended Telomeres in ku80 Mutant Cells Do Not Result treated with the indicated amounts (units) of Bal31 endonuclease, di- from Chromosome Fusion or Circularization gested with MboI, and analyzed by DNA gel blot hybridization with the telomere repeat probe (A). The DNA gel blot was washed off and re- Mammalian ku80 mutant cells present a 24-fold increase in probed with a subtelomeric region from the long arm of chromosome 2 telomeric fusions compared with wild-type cells (Samper et al., (B), which specifically detects the telomeric MboI fragment of this chro- 2000). Therefore, we asked whether the elongated telomeric re- mosome arm. ku80 and wild-type DNA were equally sensitive to Bal31 peats we observed in the Arabidopsis ku80 mutant cells could treatment.

Arabidopsis ku80 Telomeres 785

consistent with the absence of observable chromosome fusions in the male meiotic cells that we analyzed (data not shown). As mentioned above, this observation is in contrast to the mammalian ku80 phenotype of chromosome fusion and genome instability (a sixfold increase in broken chromatids and chromosome fragments in ku80 cells compared with the wild type). Furthermore, this phenotype is associated with severe infertility in ku80 mice. Arabidopsis ku80 plants were fertile at least until mutant generation 4, and we observed no cytological aberrations in male meiosis in these plants (data not shown).

The Ku80 Protein Provides a Negative Signal to Telomerase

Two mechanisms might explain the new telomeric repeat addition in ku80 mutant cells: direct addition by the telomerase and recombination between telomeric sequences (alternative lengthening of telomeres [ALT] pathway). The Ku80 protein could function as a regulator of the ALT pathway by preventing recombination between telomeres. Alternatively, Ku80 could directly regulate telomerase activity or modulate its access to the telomeres. To distinguish between these two hypotheses, we generated double-mutant ku80 attert plants by crossing the ku80 mutant with the telomerase mutant attert (Fitzgerald et al., 1999) (a kind gift of T. McKnight and D. Shippen, Texas A&M University, College Station, TX). Genotypes of F2 progeny of this Figure 4. Long Telomeres in ku80 Require Telomerase. cross were verified by DNA gel blot analysis, and wild-type, DNA gel blot analysis of telomere length using the telomeric repeat probe ku80 mutant, attert mutant, and ku80 attert double-mutant (A) and the chromosome II subtelomeric probe (B). MboI-digested DNA plants were identified. These plants are labeled G1 (for genera- from wild-type (KT), attert mutant (Kt), ku80 mutant (kT), and tion 1) in Figure 4. Progeny (selfed) of the plants are labeled G2 the ku80 attert double mutant (kt) plants was analyzed. Data from (for generation 2) in Figure 4. two subsequent generations of plants (G1 and G2) are shown. We expect the appearance of extended telomeres in the double mutant if a telomerase-independent recombination mechanism is involved in the generation of the ku80 telomeres. Telomere length was measured by DNA gel blot analysis of modified TRAP assay was used to measure telomerase activity MboI-digested genomic DNA using the telomere repeat as a in vitro in total protein extracts of wild-type and ku80 mutant probe. As expected, Figure 4A shows longer telomeres for the callus culture cells (see Methods). No telomere elongation ku80 single mutant and shorter telomeres for the attert single was detected in the absence of protein extracts (Figure 5, mutant compared with those for the wild-type plants. Telo- lane 5) or when the extracts were pretreated with RNaseA to meres present in the double mutant and in the single attert mu- inactivate the telomerase enzyme (Figure 5, lanes 1 and 2). tant were much shorter than those in both the wild type and the Protein extracts from both wild-type and ku80 mutant cells ku80 single mutants. Furthermore, both the attert and ku80 tel- showed similar levels of telomerase activity (Figure 5, lanes 3 omerase double-mutant telomeres were shorter in the subse- and 4). Thus, we were unable to detect major changes in tel- quent generation. We also checked the length dynamics of one omerase activity per se in this in vitro test. However, in vivo particular telomere in these mutants. Figure 4B shows the deregulation of telomerase activity in the absence of the Ku80 same DNA gel blot filter reprobed with a subtelomeric probe protein cannot be excluded. specific for the long arm of chromosome 2. These results confirmed that ku80 attert double-mutant plants presented shorter DISCUSSION telomeres for chromosome 2 compared with the ku80 single mutant plant or the wild type. Interestingly, an accelerated rate of We have described the role of the Arabidopsis Ku80 protein in telomere shortening was observed in the double mutant com- telomere homeostasis by studying an Arabidopsis ku80 mutant pared with the attert single mutant, indicating that the Ku80 pro- that carries a T-DNA insertion in the KU80 gene. Mutant plants tein has another, positive influence on telomere length in the ab- presented longer telomeres than wild-type plants. This pheno- sence of telomerase. type is similar to that reported recently for an Arabidopsis ku70 Given the telomerase dependence of the extended telomeres mutant (Bundock et al., 2002; Riha et al., 2002). A progressive in ku80 cells, we performed an in vitro telomerase assay to increase in the number of telomeric repeats was detected in check for an increase in telomerase activity in these cells. The ku80 mutants, which in callus culture reached a maximum

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length and stabilized. The Bal31 exonuclease sensitivity of these long telomeres indicates that they do not result from end- to-end chromosome fusion or circularization. This finding was confirmed by cytological analysis of male meiotic cells from generation-4 ku80 mutant plants. Additional generations of mutant plants will be monitored for cytological aberrations. Two mechanisms could be involved in the generation of the extended telomeric repeats: recombination and direct addition of telomeric repeats by the telomerase. Analysis of Arabi- dopsis ku80 attert double-mutant plants demonstrated that telomere elongation in Ku80-deficient plants was telomerase dependent. Furthermore, Ku80 deficiency did not significantly affect telomerase activity measured in cell extracts, suggesting that Ku80 acts a negative telomere length regulator at individual chromosomes in plants. The essential role of the Ku70/Ku80 heterodimer in telomere metabolism has been demonstrated in several organisms. These data indicate species-specific mechanisms for Ku protein function in telomeres. Saccharomyces cerevisiae and Schizosaccharomyces pombe cells deficient in Ku70 present shortened but stable telomeres (Boulton and Jackson, 1996; Porter et al., 1996; Baumann and Cech, 2000). In neither case are chromosome fusions observed. Interestingly, in S. cerevisiae, simultaneous deletion of the telomerase catalytic subunit and Ku70 or Ku80 causes lethality (Gravel et al., 1998; Nugent et al., 1998). By contrast, ku70 telomerase double-mutant fission yeast cells senesce, but a large number of survivors are Figure 5. Telomerase Activity in Ku/ Mutant Cells. generated. These surviving cells present circularization of all three chromosomes, as is observed for telomerase-deficient Protein extracts were prepared from wild-type () and homozygous (k) survivors in this organism (Nakamura et al., 1998; Baumann ku80 mutant cells. The fifth lane ( ) contained no protein extract. Tel- and Cech, 2000). Conflicting results concerning telomeres have omerase activity was measured by the modified TRAP assay as described in Methods. Extracts were pretreated () or not () with RNase. been obtained in ku80 knockout mice: in one study, extended Band intensity was quantified by phosphorimager analysis and is shown telomeres were observed, whereas a second study showed a graphically at right for the wild type () and the ku80 mutant (ku80). decrease in telomeric repeats (Samper et al., 2000; d’Adda di Fagagna et al., 2001). Nevertheless, a high frequency of end- to-end fusions was found in both studies. These results suggest a role for the Ku80 protein in telomere end capping. ku70 study of telomerase-deficient cells. TRF1 and TRF2 are double- disruption in chicken DT40 cell lines has no effect on telomere stranded TTAGGG-repeat binding proteins. It has been pro- length and does not result in chromosome fusions (Wei et al., posed that they control telomere length in cis by inhibiting the 2002). Recently, it was shown that Arabidopsis plants carrying action of telomerase. In fact, both proteins are needed for the a T-DNA insertion in the KU70 gene have extended telomeres, generation and maintenance of the T-loop (Bianchi et al., 1997, and up to mutant generation 2, chromosome fusions are not ob- 1999; Griffith et al., 1998, 1999; Stansel et al., 2001). In the served (Riha et al., 2002). Here, we show that Ku80-deficient Ara- T-loop, the 3 end DNA strand overhang required for telomer- bidopsis presented an elongation of telomeres, also without chro- ase action is sequestered into the duplex part of the telomeric mosome fusions. Thus, it appears that in all organisms studied to repeat tract. TIN2 interacts with TRF1 and has been suggested date, with the exception of chicken DT40 cells, the Ku80 protein to stabilize the T-loop (Kim et al., 1999). The Ku80 protein inter- plays a role in telomere length homeostasis, with an additional acts with TRF1 and TRF2 and may contribute to the stabiliza- role in mammalian cells to protect telomeres from fusion. tion of the T-loop structure and control the action of telomerase Telomeres have a species-specific length that is constant (Hsu et al., 2000; Song et al., 2000). over generations, implying tight regulatory mechanisms to Alternatively, the role of the Ku80 protein could be to sup- measure and modulate the length of the telomeric repeat at in- press the accessibility to telomeres of the homologous recom- dividual chromosome ends. In mammalian cells, the TRF1, bination machinery. S. cerevisiae and S. pombe ku mutants TRF2, and TIN2 telomere binding proteins have been sug- show an increase in subtelomeric and telomeric recombination, gested to function in the negative regulation of telomerase ac- notwithstanding the presence of shorter telomeres. S. cerevisiae tion (Kim et al., 1999; Smogorzewska et al., 2000). Disruption of and mammalian cells lacking telomerase activity present heter- the function of any of these proteins gives rise to the generation ogeneous telomere length as a result of a telomerase-indepen- of longer telomeres. However, direct evidence that the ex- dent pathway of telomere maintenance that involves recombi- tended telomeres are telomerase dependent must await the nation (Lundblad and Blackburn, 1993; Bryan et al., 1995; Teng Arabidopsis ku80 Telomeres 787

and Zakian, 1999; Dunham et al., 2000). A recent analysis of arm. Specific details have been described previously (Gallego and yeast ku telomerase double mutants by Tsai et al. (2002) has White, 2001). shown that the Ku complex plays an additional role in protecting telomeres and in generating survivors in telomerase mu- Telomerase Repeat Amplification Protocol Assay tants. Thus, Ku80 protein action in telomere homeostasis involves blocking telomerase activity as well as stabilizing the Whole cell extracts from suspension culture cells were prepared according to Fitzgerald et al. (1996). The telomerase TRAP assay was per- telomeres and facilitating the access of the homologous re- formed according to the protocol of Szatmari and Aradi (2001) with mod- combination proteins to telomeres. Both longer and shorter ifications. The following oligonucleotides were used: forward primer telomeres have been described in mouse ku80 mutants, and o177 (5-CACTATCGACTACGCGATCGG-3), tagging primer o278 (5- telomere lengthening has been shown to be telomerase depen- GATCTCGAGCTCGATATCGGATCCCCTAAACCCTAAAGG-3), and tag dent (Espejel et al., 2002). primer o274 (5-GATCTCGAGCTCGATATCGGATC-3). The assay was The gradual elongation and subsequent stabilization of telo- performed as follows: 0.5 g of extract and 1 L (10 pmol) of primer meres we observed in ku80 mutant plant cells suggests a tel- o177 were added to 40 L of assay buffer (50 mM Tris acetate, pH 8.3, omerase-mediated, rather than a recombination (ALT), mecha- 5 mM MgCl2, 10 mM EGTA, 1 mM DTT, 50 mM potassium glutamate, nism in Arabidopsis. We have confirmed this by showing that 0.1% Triton X-100, 1 mM spermidine, 50 M each deoxynucleotide the appearance of long telomeres in Ku80-deficient Arabidop- triphosphate, and 100 ng/ L BSA). This mixture was incubated at 22 C for 30 min and then heated to 90C for 3 min. During the 90C incubation, sis depends on the presence of telomerase. An alternative re- 6.5 L of PCR mix (15 pmol of primer o177, 25 pmol of primer o274, 0.5 combination-based (unequal exchange) and telomerase-depen- pmol of primer o278, 5 Ci of -32P-dCTP, and 0.5 units of Taq DNA dent mechanism also could explain these results; however, polymerase [Qiagen, Valencia, CA]) was added. PCR then was per- such a mechanism would not be expected to lead to the ob- formed as follows: 2 cycles of 94C for 30 s, 55C for 60 s, and 72C for served stabilization of the elongated telomere length (Figure 2). 90 s, followed by 27 cycles of 94C for 30 s, 63C for 30 s, and 72C for Interestingly, the ku telomerase double-mutant plants have 30 s, incubation at 72C for 90 s, and cooling to 4C. Products were sep- even shorter telomeres than the single telomerase mutants. arated on 6% denaturing acrylamide sequencing gels, and the gels were This synergism between the ku80 and telomerase mutations dried and autoradiographed with a phosphorimager (Bio-Rad Personal FX). indicates a role for the Ku80 protein in telomere stability in addition to its role as a negative regulator of telomerase ac- Bal31 Sensitivity Assay tion. As mentioned above, Tsai et al. (2002) recently reported a role for the Ku complex in telomere protection and survivor Total genomic DNA (1 g) was treated with different concentrations of Bal31 exonuclease (New England Biolabs, Beverly, MA) for 30 min at generation in yeast. A similar role(s) in Arabidopsis would ex- 37C in the manufacturer’s buffer in a volume of 50 L. After the addition plain this synergistic effect. of EGTA to 20 mM and 10 min of incubation at 65C, the DNA samples Thus, the Arabidopsis Ku80 protein could directly regulate were extracted in phenol/chloroform and precipitated in sodium acetate/ the action of telomerase at telomeres or possibly regulate other ethanol. MboI digestion and DNA gel blot analysis were as described proteins essential for telomere homeostasis as well as play a above. role in telomere end protection or stability. Full understanding Upon request, all novel materials described in this article will be made of Ku80’s role in telomere metabolism in plant cells will require available in a timely manner for noncommercial research purposes. the identification of the other protein partners present at the telomeres. Accession Number

The GenBank accession number for the AtKU80 clone is AF283758. METHODS

Plant Growth and Callus Induction ACKNOWLEDGMENTS Arabidopsis thaliana seeds were sown directly into damp compost, and plants were grown in a greenhouse under standard conditions. Callus cultures were derived from leaves and maintained as described previ- We thank Dorothy Shippen and Tom McKnight for the attert mutant. This ously (Gallego and White, 2001). work was financed partly by grants from the Commissariat à l’Energie Atomique France (Laboratoire de Recherche Conventionné Commissar- iat à l’Energie Atomique No. 19V) and the European Union (Contract QLG2-CT-2001-01397). DNA Isolation and DNA Gel Blot Analysis

DNA was prepared from plants or callus cells as described previously (Gallego and White, 2001). TRF analysis was performed in MboI-digested Received October 15, 2002; accepted December 5, 2002. DNA (0.5 to 1 g), which was separated by electrophoresis on 0.8% agarose/Tris-borate-EDTA gels and blotted to positively charged nylon membranes (Hybond N ; Amersham). The subtelomeric chromosome II REFERENCES probe was labeled with -32P-dCTP using the Prime It II kit (Stratagene), and the telomeric repeat probe was 5 labeled using T4 polynucleotide kinase and -32P-ATP. The chromosome 2 long-arm subtelomeric probe Bailey, S.M., Meyne, J., Chen, D.J., Kurimasa, A., Li, G.C., Lehnert, specifically detects the telomeric MboI fragment of this chromosome B.E., and Goodwin, E.H. (1999). DNA double-strand break repair 788 The Plant Cell

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