ELG1, a Regulator of Genome Stability, Has a Role in Telomere Length Regulation and in Silencing

ELG1, a regulator of genome stability, has a role in telomere length regulation and in silencing

Sarit Smolikov*, Yuval Mazor, and Anat Krauskopf†

Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israel

Communicated by Elizabeth Blackburn, University of California, San Francisco, CA, December 6, 2003 (received for review August 20, 2003) Telomeres, the natural ends of eukaryotic chromosomes, prevent link between telomere length regulation and the DNA replica- the loss of chromosomal sequences and preclude their recognition tion machinery (19). This interaction might underline the sig- as broken DNA. Telomere length is kept under strict boundaries by nificance of Cdc13p in recruiting both telomerase and DNA the action of various proteins, some with negative and others with polymerase to the telomere. One of the models raised (19) positive effects on telomere length. Recently, data have been suggested that proteins involved in DNA replication might accumulating to support a role for DNA replication in the control compete with telomerase for the telomeric substrate. of telomere length, although through a currently poorly under- When the replication machinery reaches the end of a linear stood mechanism. Elg1p, a replication factor C (RFC)-like protein of DNA molecule, on its lagging strand, it might interact with the yeast, contributes to genome stability through a putative replica- telomerase enzymatic complex. In agreement with this finding, tion-associated function. Here, we show that Elg1p participates in some of the genes involved in DNA replication also influence negative control of telomere length and in telomeric silencing telomere length regulation. Pol␣, Pol␦, and DNA primase are through a replication-mediated pathway. We show that the telo- required for telomere addition. It was suggested that in their meric function of Elg1 is independent of recombination and com- absence telomerase is inactive (20). Mutations in the large pletely dependent on an active telomerase. Additionally, this subunit of replication factor C (RFC) (Rfc1p͞Cdc44p) and of function depends on yKu and DNA polymerase. We discuss alter- DNA polymerase &␣␣␹␷␶␧ (Pol␣͞Cdc17p) cause telomere elon- native models to explain how Elg1p affects telomere length. gation (21, 22). The RFC complex is required for DNA replication and repair. It recognizes primer-template structures and DNA replication ͉ replication factor C ͉ Saccharomyces cerevisiae acts as a ‘‘clamp-loader’’ to recruit proliferating cell nuclear antigen (PCNA), the DNA replication processivity factor (23). ␣ eplication of a linear DNA molecule is carried out by DNA Mutations in Pol and in PCNA are also associated with loss of Rpolymerases and additional proteins that duplicate the DNA telomeric silencing (24, 25). by moving coordinately on both the lagging and leading strands Elg1p exhibits sequence similarity to Rfc1p and interacts with (1). DNA attrition at the ends of chromosomes due to the ‘‘end Rfc2 to -5 proteins (but not with Rfc1p itself). These facts may replication problem’’ is avoided by the action of the specialized point to the existence of an alternative RFC complex, in which reverse transcriptase, called telomerase (2, 3). Telomerase adds Rfc1p is replaced by Elg1p (26, 27). At least two other similar RFC complexes exist in yeast; in these complexes, Rfc1p is G-rich DNA repeats termed telomeres onto the ends of chro- replaced by either Ctf18p or Rad24p (28, 29). Deletion of CTF18 mosomes. In addition to their role in preventing loss of chro- causes telomere shortening (30) whereas a RAD24 deletion does mosomal sequences, telomeres serve as a boarding pad for a not affect telomere length. ELG1 was identified recently as a multiprotein complex that is implicated in several important gene whose deletion causes increased levels of recombination. cellular functions. Among these functions is capping telomeres, Mutations in ELG1 exhibit impaired growth or lethality in thus avoiding the recognition of the natural ends of chromo- combination with mutations in genes involved in homologous somes as broken DNA. This capping in turn prevents cell cycle recombination. These findings raised the possibility that ELG1 arrest and chromosomal fusions, allowing proper chromosomal may act to suppress replication-related DNA damage, thereby segregation (4). functioning as a keeper of genome stability (26, 27). Many proteins, most of which are part of the telomeric Here, we show that deletion of ELG1 causes telomere elon- complex, regulate telomere length in both positive and negative gation, unraveling a role for Elg1p in limiting telomere length. fashions (5). The best-studied pathway of telomerase-negative We further characterize the telomeric pathway in which Elg1p regulation is the Rap1p pathway in yeast. Rap1p is bound participates and suggest a model to explain the relation between throughout the telomeric sequence and interacts with two pro- the roles of Elg1p in DNA replication and in determination of teins, Rif1p and Rif2p (6, 7). The resulting complex is thought telomere length. to prevent telomerase from accessing the telomeric DNA substrate by a yet unknown mechanism. Additional factors were Materials and Methods shown to be involved in telomere length regulation. Tel1p and Plasmid Construction. pELG1 was constructed by cloning a 2.3-kb the yKu70p-yKu80p heterodimer act as positive regulators of BamHI-PstlI PCR-amplified fragment of ELG1 in the BamHI- telomerase (8–10). Tel1p is proposed to act by recruiting the PstlI site of pCM189 (31). MRX (Mer11͞Rad50͞Xrs2) complex (11). The yKu70p͞yKu80p heterodimer influences telomere length by protecting telomere Strain Construction. All deletion strains were of BY4741 back- ends from degradation. It also has additional functions in ground. Strains containing the temperature-sensitive alleles telomeric silencing (through recruiting Sir proteins to the telo- cdc17-1 (22) and cdc44-1 (32) were transferred to the BY4741 meres), telomeric nuclear localization, and telomere replication timing (12–15). Cdc13p binds the telomeric single strand, re- cruits telomerase to the telomere, and prevents degradation (16, Abbreviations: Pol, polymerase; RFC, replication factor C; PCNA, proliferating cell nuclear 17). By these functions, Cdc13p acts as a positive regulator of antigen; YPD, yeast extract͞peptone͞dextrose; SD-Ura, synthetic dextrose medium lacking telomere length. In addition, Cdc13p also regulates telomerase uracil. negatively by binding Stn1p, which prevents telomerase recruit- *To whom correspondence should be addressed. E-mail: [email protected]. ment to the telomeres (18). In addition Cdc13p interact physi- †Deceased October 3, 2003. cally and genetically with polymerase (Pol) ␣, thus providing a © 2004 by The National Academy of Sciences of the USA

1656–1661 ͉ PNAS ͉ February 10, 2004 ͉ vol. 101 ͉ no. 6 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307796100 Downloaded by guest on September 24, 2021 Diploids heterozygous for ELG1 and a second gene were dissected, and tetra-type tetrads were selected. The genotypes of the spores were confirmed by PCR. A minimum of two tetrads from each cross was analyzed. All strains were grown at 30°C and on yeast extract͞peptone͞dextrose (YPD) media, unless stated differently. Strains transformed with pELG1 were grown on synthetic dextrose medium lacking uracil (SD-Ura) media to avoid plasmid loss.

Silencing Tests. Cells were grown on YPD liquid media supplemented with adenine (2 mg͞ml) for 1 day. Cultures were plated on YPD plates and incubated until development of red color.

Southern Blots. Genomic DNA was prepared from saturated cultures by using a modified version of the zymolase method and hybridized as described (34). Saccharomyces cerevisiae-specific telomeric probe was prepared by phosphorylation with T4 polynucleotide kinase of a telomeric G-strand oligonucleotide. Hybridizations (1 h) and washes (total 20 min) were performed at 45°C. The G-strand oligonucleotide was 5Ј-TGGGTGT- GGGTGTGGGTGTGGGTG-3Ј; the C strand 12 oligonucleotide was 5Ј-CACCCACACCCACACCCACACCCA-3Ј. Fig. 1. Elongation of telomeres in ⌬elg1 depends on telomerase, but not on recombination. Shown is Southern blot analysis with an S. cerevisiae-speciﬁc Results telomeric probe. (A) Genomic DNA was prepared from spore derivatives dissected from ELG1͞⌬elg1 RAD52͞⌬rad52 tetrads. Lane 1, ELG1 RAD52; lane We have performed a systematic screen of the entire collection 2, ⌬elg1 RAD52; lane 3, ELG1 ⌬rad52 single mutants; lane 4, ⌬elg1 ⌬rad52 of viable S. cerevisiae haploid gene deletion mutants for genes double mutant. (B) Genomic DNA was prepared from spore derivatives dis- whose deletion causes telomeric shortening or elongation, with sected from ELG1͞⌬elg1 EST2͞⌬est2 tetrads. Lane 1, ⌬elg1 ⌬est2 double the goal of obtaining a full genomic view of genes involved in mutant; lane 2, ⌬elg1 EST2; lane 3, ELG1 EST2; lane 4, ELG1 ⌬est2. telomere metabolism (A.K. and M. McEachern, unpublished results). Among the deletion strains that exhibited a deviation from the wild-type telomeric pattern, we identified ⌬elg1 as one genetic background by backcrosses. The strain containing the of the strains with elongated telomeres. ADE2 telomeric gene was created by three backcrosses of the To confirm that the telomere elongation phenotype was UCC41 strain provided by D. Gottschling (33). All deletion indeed the result of deletion of the ELG1 gene, we performed strains used contain replacement of the gene ORF by a KanMX several genetic tests. The first was a tetrad analysis test, which cassette, with the exception of CTF18, which is replaced by HygB. showed that the telomeric phenotype indeed cosegregated with GENETICS

Fig. 2. Elongation of telomeres in ⌬elg1 depends on KU70, but not on RIF2, RIF1,orTEL1. Southern blot analysis with an S. cerevisiae-speciﬁc telomeric probe was done. (A) Genomic DNA was prepared from spore derivatives dissected from ELG1͞⌬elg1 RIF2͞⌬rif2 tetrads. Lane 1, ⌬elg1RIF2; lane 2, ELG1 ⌬rif2; lane 3, ELG1 RIF2; lane 4, ⌬elg1 ⌬rif2 double mutant. (B) Genomic DNA was prepared from spore derivatives dissected from ELG1͞⌬elg1 RIF1͞⌬rif1 tetrads. Lane 1, ELG1 RIF1; lane 2, ⌬elg1RIF1; lane 3, ELG1 ⌬rif1 double mutant; lane 4, ⌬elg1 ⌬rif1.(C) Genomic DNA was prepared from spore derivatives dissected from ELG1͞⌬elg1 TEL1͞⌬tel1 tetrads. Lane 1, ELG1 TEL1; lane 2, ELG1 ⌬tel1; lane 3, ⌬elg1TEL1; lane 4, ⌬elg1 ⌬te1l double mutant. (D) Genomic DNA was prepared from spore derivatives dissected from ELG1͞⌬elg1 YKU70͞⌬yku70 tetrads. Lane 1, ELG1 YKU70; lane 2, ⌬elg1 YKU70; lane 3, ⌬elg1 ⌬yku70 double mutant; lane 4, ELG1 ⌬yku70.

Smolikov et al. PNAS ͉ February 10, 2004 ͉ vol. 101 ͉ no. 6 ͉ 1657 Downloaded by guest on September 24, 2021 Fig. 3. ELG1 operates in telomere elongation in the same genetic pathway as POL␣, but in a distinct genetic pathway from CTF18 and RFC1. DNA was analyzed by Southern blot hybridized with an S. cerevisiae-speciﬁc telomeric probe. (A) Genomic DNA was prepared from spore derivatives dissected from ELG1͞⌬elg1 CDC44͞cdc44-1 tetrads. Lane 1, ELG1 CDC44; lane 2, ELG1 cdc44-1; lane 3, ⌬elg1 CDC44; lane 4, ⌬elg1 cdc44-1 double mutant. (B) Genomic DNA was prepared from spore derivatives dissected from ELG1͞⌬elg1 CTF18͞⌬ctf18 tetrads. Lane 1, ELG1 CTF18; lane 2, ELG1͞⌬ctf18; lane 3, ⌬elg1͞⌬ctf18 double mutant; lane 4, ⌬elg1 CTF18.(C) Genomic DNA was prepared from spore derivatives dissected from ELG1͞⌬elg1 CDC17͞cdc17-1 tetrads. Spores 1–4 were grown at 25°C, and spores 5–8 were grown at 30°C. Lane 1, ELG1 CDC17; lane 2, ⌬elg1 cdc17-1 double mutant; lane 3, ⌬elg1 CDC17; lane 4, ELG1 cdc17-1; lane 5, ELG1 CDC17; lane 6, ELG1 cdc17-1; lane 7, ⌬elg1 cdc17-1 double mutant; lane 8, ⌬elg1 CDC17.

the kanamycin resistance phenotype conferred by the deletion were involved in telomere elongation of ⌬elg1, we would expect marker. The phenotype appeared by the first passage after the deletion of EST2 to suppress this phenotype. Indeed, as sporulation but reached its full potential by the fifth passage in shown in Fig. 1B, lane 1, deletion of EST2 prevented telomere all spore clones tested (data not shown). As a second test, we elongation of ⌬elg1. These results show that telomere elongation expressed ELG1 from a plasmid in a ⌬elg1 strain. This expression in the ⌬elg1 mutant is telomerase-dependent. resulted in telomeres shortening back to the wild-type length As mentioned above, Rif2p and Rif1p were found to regulate (data not shown). These two independent tests confirmed that telomerase negatively by binding to the C-terminal domain of disruption of ELG1 was responsible for the observed telomere Rap1p. Deletion of either gene results in telomere elongation length elongation in the ⌬elg1 strain. (7). Therefore, it was possible that ELG1 acts in the same genetic Our aim was to characterize the cellular pathway in which pathway as RAP1 and the associated factors RIF1 and RIF2.As ELG1 acts in preventing telomere elongation. As mentioned, shown in Fig. 2A, lane 4, deletion of both RIF2 and ELG1 ELG1 was identified as a gene responsible for suppression of resulted in telomere lengthening that was more severe than that various types of chromosomal recombination (26, 27). observed in each of the single mutant (Fig. 2A, lanes 1 and 2). Therefore, one possibility was that deletion of ELG1 pro- Double deletion of ELG1 and RIF1 also seemed to have a similar moted recombination between telomeric repeats, thereby result- effect on telomere length although, because the effects of ing in elongated telomeres. We thus tested the involvement of deletion of RIF1 were so severe, the additive effect of the double RAD52, a key component of the homologous recombination deletion on telomere length was less clear-cut (Fig. 2B, etc.). pathway, in telomere elongation of the ⌬elg1 strain. As shown, Therefore, we conclude that the negative regulation of telomere ⌬elg1 ⌬rad52 (Fig. 1A, lane 4) had the same telomere elongation length by ELG1 and RIF2, and probably RIF1, is carried out phenotype as the ⌬elg1 strain (Fig. 1A, lane 2), demonstrating through distinct pathways. that telomere elongation in the ⌬elg1 strain was not dependent Two additional genes known to affect telomere length are on recombination. YKU70 and TEL1. Deletion of either gene results in dramatic To further understand the mechanism underlying the telo- telomere shortening, but the two genes were mapped to two mere elongation phenotype caused by the deletion of ELG1,we distinct pathways of telomerase positive regulation (10). To test tested the involvement of several genes mapped to different whether ELG1 acts at one of these genetic pathways, the telomeric length regulation pathways: EST2, RIF2, RIF1, YKU70, telomeric phenotypes of the corresponding double mutants were and TEL1. tested. As shown in Fig. 2C, lane 4, telomere length of the ⌬tel1 To test whether telomere elongation in the ⌬elg1 strain ⌬elg1 mutant exhibited an intermediate phenotype, resembling involved the activity of telomerase, we examined the telomeric that of the wild-type strain, demonstrating that TEL1 and ELG1 phenotype of the double mutant ⌬elg1 ⌬est2. EST2 encodes the act in distinct pathways affecting telomere length. In contrast, catalytic subunit of telomerase (35). Therefore, if telomerase telomere length of the double mutant ⌬yku70 ⌬elg1 (Fig. 2D,

1658 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307796100 Smolikov et al. Downloaded by guest on September 24, 2021 lane 3) resembled that of the single mutant ⌬yku70 (Fig. 2D, lane 4). This finding shows that ELG1 and YKU70 act in the same pathway of telomere length regulation and that YKU70 is required for the elongated telomere phenotype of the ⌬elg1 mutant. If ELG1 participated in an alternative RFC complex, distinct from those described for RFC1, RAD24, and CTF18,itis expected that its effect on telomere length might also be carried out through a distinct genetic pathway. As seen in Fig. 3A, lane 4, the phenotype of the double mutant cdc44-1 ⌬elg1 is much more severe than the phenotypes of either single mutant (lanes 2 and 3). The telomeric length of the double mutant ⌬elg1 ⌬ctf18 (Fig. 3B, lane 3) is shorter than that of the single ⌬elg1 mutant (Fig. 3B, lane 4) and longer than that of the single ⌬ctf18 mutant (Fig. 3B, lane 2). Although deletion of RAD24 does not affect telomere length, we tested whether it affects telomere length in combination with ⌬elg1. The telomeric length of the double mutant ⌬elg1 ⌬rad24 resembled that of the single mutant ⌬elg1 (data not shown). These experiments demonstrate that the function of Elg1p is distinct from that of the other alternative RFC genes in relation to telomere length. We also tested the genetic interaction between the cdc17-1 allele of Pol␣ and a deletion of ELG1.As shown in Fig. 3C, telomere length of the double mutant cdc17-1 ⌬elg1 was similar to that of the single cdc17-1 strain at 25°C and at 30°C (Fig. 3C). Therefore, we conclude that ELG1 and CDC17 regulate telomere length negatively through the same genetic pathway. Mutations in genes involved in replication of the lagging strand (Pol␣, RAD27) cause an increase in the amount of telomeric single-stranded DNA (ssDNA) (24, 36). We examined the telomeric ssDNA in rad24, ctf18, rfc1, and elg1 mutants and found no accumulation of ssDNA (data not shown). Therefore, there is no direct correlation between the telomere length phenotype of RFC-like proteins, including Elg1p, and accumulation of telomeric ssDNA. Genes involved in telomere length regulation often influence Fig. 4. Deletion of ELG1 elevates silencing at an ADE2 gene placed near the the structure of the telomeric heterochromatin, and these telomere in S. cerevisiae.(A) Complementation of the elevated telomeric changes are sometimes associated with an increase or decrease silencing phenotype was tested in the indicated strains. Strains containing the in the transcription of genes placed adjacent to the telomere ⌬elg::KanMX deletion allele or the ELG1 allele were transformed with control (13, 33, 37). (pCM189) or pELG1 plasmids. The strains were grown on YPD media supple- Telomere position effect can be easily monitored by placing mented with adenine and plated on SD-Ura plates. (B) The uncoupling of the ADE2 gene near the telomere of chromosome VII (33). We silencing and length phenotype of ⌬elg1 was tested by picking separately ϩ Ϫ generated a heterozygous strain (elg1͞ELG1) containing a sub- Ura (lane 4) and Ura (lane 3) colonies for extraction of genomic DNA. DNA was analyzed by Southern blot hybridized with an S. cerevisiae-speciﬁc telo- telomeric ADE2 gene. Spore derivatives of ELG1 genotype ⌬ showed normal silencing at the ADE2 locus whereas spore meric probe. Telomeric length was compared with elg::KanMX strain (lane 1) ⌬ and ELG1 (lane 2). (C) Silencing was tested in a strain containing the derivatives of the elg1 genotype exhibited a substantial eleva- ⌬elg::KanMX deletion allele and pELG1 plasmid. This strain was transferred tion of silencing, evident from the pink coloration of the colonies from selective media (SD-Ura) to nonselective media (YPD). Colonies were (Fig. 4A). This phenotype can be suppressed by a plasmid replica-plated on SD-Ura plates. (D) Genomic DNA was prepared from tetrad harboring ELG1 (Fig. 4A), demonstrating that the absence of created by the dissection of an ELG1͞⌬elg1 TEL1͞⌬tel1 diploid with an ADE2 ELG1 is the cause for the appearance of pink colonies. telomeric gene. Spores with all gene combinations (ELG1, ⌬elg1, ⌬tel1, and GENETICS We further examined whether the elevation in telomere ⌬elg1 ⌬tel1) and a telomeric ADE2 gene were grown on YPD liquid media position effect arises directly from the elongation in telomere supplemented with adenine and plated on a YPD plate. length. We tested the telomeric silencing phenotype of the ⌬elg1 strain immediately after loss of the pELG1 plasmid, while its telomeres were still only moderately elongated (Fig. 4B), and activity of silencing factors (38), we tested their involvement in elevation of silencing in the ⌬elg1 strain. As expected, telo- found that, even when telomere length is close to wild-type meric silencing of the ⌬elg1 strain depended on SIR2,an levels, elevation in telomere position effect is apparent (Fig. 4C). ⌬ ⌬ essential component of the telomeric silencing complex (Fig. In addition, we observed that tel1 elg1 exhibited an elevated 5A). As mentioned, yKu70p is essential for telomeric silencing level of telomeric silencing that resembles that of the single ⌬ ⌬ (12). Telomeric silencing of the elg1 strain was also depen- mutant elg1 (Fig. 4D), despite the fact that its telomere length dent on YKU70 (Fig. 5B). In summary, elevation of silencing resembles that of the wild-type strain (Fig. 2C). The single in the ⌬elg1 strain depended on the essential components of ⌬ mutant tel1 exhibited telomeric silencing levels that resembled the telomeric silencing complex in the same manner as normal that of the wild-type strain, as expected. telomeric silencing. Therefore, we conclude that the elevation of telomeric silencing in the ⌬elg1 strain was probably due to the lack of Discussion Elg1p in this strain and not to elongation of the telomere. The main finding of this article is that the alternative RFC Because telomeric silencing was shown to depend on the component Elg1p participates in negative control of telomere

Smolikov et al. PNAS ͉ February 10, 2004 ͉ vol. 101 ͉ no. 6 ͉ 1659 Downloaded by guest on September 24, 2021 Here, we show that deletion of an ELG1 results in an increase in telomere length and that the effect of ELG1 on telomeres is genetically unlinked from that of other RFC-like complexes. Our results point strongly to the involvement of DNA replication in the telomeric function of Elg1p. We show that, in the absence of ELG1, elevation of telomere length depends on CDC17͞Pol␣ and on YKU70. Mammalian Ku was found to interact with DNA polymerases ␣, ␦, and ␧, PCNA, and the RFC complex (43). These results may also indicate that yKu70p might be involved in DNA replication. ELG1 may operate as an alternative sliding- clamp loader that loads an additional unknown PCNA-like complex according to particular cellular needs (26, 27, 41). It is possible that one of these functions associated with ELG1 also limits telomere length and telomeric silencing.

The Role of Alternative Elg1p-RFC in Telomere Length Regulation and Telomeric Silencing. We propose two alternative models for the function of Elg1p at the telomere. The first model is based on the competition between telomerase and DNA polymerase. At the telomere, both enzymatic complexes compete for the same Fig. 5. Increased silencing at an ADE2 gene placed near the telomere in substrate, namely, telomeric DNA. According to this model, a⌬elg1 background depends on YKU70 and SIR2.(A) ELG1͞⌬elg1 SIR2͞⌬sir2 Elg1p functions to open telomeric chromatin structures that diploid with an ADE2 telomeric gene was dissected. Spores with all gene interfere with passage of the replication fork. In this manner, it combinations (ELG1, ⌬elg1, ⌬sir2, and ⌬elg1 ⌬sir2) and a telomeric ADE2 gene functions as a processivity factor for replication of the telomeric were grown on YPD liquid media supplemented with adenine and plated on region. In the absence of Elg1p, DNA polymerase is less able to a YPD plate. (B) ELG1͞⌬elg1 YKU70͞⌬yku70 diploid with an ADE2 telomeric compete with telomerase due to its reduced ability to pass gene was dissected. Spores with all gene combinations (ELG1, ⌬elg1, ⌬yku70, through the telomeric chromatic region. As a result, telomerase and ⌬elg1 ⌬yku70) and a telomeric ADE2 gene were grown on YPD liquid gains better access to its substrate, leading to the observed media supplemented with adenine and plated on a YPD plate. increased telomere length. The telomeric chromatin structure remains at the telomere, resulting in increased levels of telomeric silencing. length and telomeric silencing. We have characterized both The second model assumes that the telomeric phenotype phenotypes and discovered that the increase in telomere length observed in the absence of Elg1p is due to increased levels of observed in the absence of ELG1 requires an active telomerase DNA double-strand breaks. It has been proposed that the and does not depend on homologous recombination although it absence of Elg1p causes replication fork collapse or a defect in was previously shown that deletion of ELG1 increases DNA fork restart, resulting in the accumulation of genome-wide DNA recombination in other regions of the genome. Our study of the lesions, including double-strand breaks (26, 27, 41). At the genetic pathway in which ELG1 acts showed that ELG1 is telomere, these breaks may recruit the nonhomologous end- independent of TEL1, RIF1,orRIF2. joining protein complex, including yKu70p. Once the telomere In contrast, we show that the effect of Elg1p on telomere associates with more yKu70p, telomerase is recruited more length depends on yKu70p, a component of the NHEJ pathway efficiently to the telomere, hence the observed increased telo- and on Pol␣. We have also found that the increase in telomeric mere length in ⌬elg1 strains. The yKu complex has a well silencing seen in the absence of ELG1 depended on yKu70p, documented role in recruitment of the Sir proteins to the which is known to recruit the Sir complex to the telomeres (15, telomeres (39). It is therefore possible that, in the absence of 39), and on Sir2p, a major component of the telomeric silencing Elg1p, excess of yKu complex at the telomere end leads to the complex (40). Elg1p probably affects silencing directly because increased telomeric silencing through more efficient recruitment we have shown that the telomere position effect enhancement in of the Sir complex. ⌬elg1 strains is not a consequence of telomere elongation. Further studies are required to determine whether ELG1 affects telomere length through a direct role in replication (e.g., ELG1 and DNA Replication. Mutations in genes that participate in by interacting with PCNA͞PCNA-like complex). Alternatively, various aspects of DNA replication result in increased telomere it may play a more indirect role, namely through effects on length (21, 22). It was previously shown that mutations in RFC1 chromatin and thereby accessibility of the opposing enzymatic and CTF18,anRFC1-like gene, affect telomere length (21, 30). machineries working at the telomere. Rad24p, another Rfc1-like protein, which was shown to participate in a telomere checkpoint in the absence of telomerase (40), This article is dedicated to the children of Anat Krauskopf, Yotam and does not affect telomere length. Therefore, not all RFC-like Merav. We thank all members of the Krauskopf laboratory and Martin complexes are associated with telomere length control. Bio- Kupiec for scientific ideas and for critically reading the manuscript. This chemical studies guided by sequence analysis have shown that work was supported by the Israeli Cancer Foundation and the Israeli Elg1p is part of an alternative RFC complex (26, 27, 41, 42). Science Foundation.

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