Mre11-Rad50-Xrs2 Research Articles

DSB-induced oxidative stress: Uncovering crosstalk between DNA damage response and cellular metabolism

While that ROS causes DNA damage is well documented, there has been limited investigation into whether DNA damages and their repair processes can conversely induce oxidative stress. By generating a site-specific DNA double strand break (DSB) via I-SceI endonuclease expression in S. cerevisiae without damaging other cellular components, this study demonstrated that DNA repair does trigger oxidative stress. Deleting genes participating in the initiation of the resection step of homologous recombination (HR), like the MRX complex, resulted in stimulation of ROS. In contrast, deleting genes acting downstream of HR resection suppressed ROS levels. Additionally, blocking non-homologous end joining (NHEJ) also suppressed ROS. Further analysis identified Rad53 as a key player that relays DNA damage signals to alter redox metabolism in an HR-specific manner. These results suggest both HR and NHEJ can drive metabolism changes and oxidative stress, with NHEJ playing a more prominent role in ROS stimulation. Further analysis revealed a correlation between DSB-induced ROS increase and enhanced activity of NADPH oxidase Yno1 and various antioxidant enzymes. Deleting the antioxidant gene SOD1 induced synthetic lethality in HR-deficient mutants like mre11Δ and rad51Δ upon DSB induction. These findings uncover a significant interplay between DNA repair mechanisms and cellular metabolism, providing insights into understanding the side effects of genotoxic therapies and potentially aiding development of more effective cancer treatment strategies.

Molecular insights into the activation of Mre11-Rad50 endonuclease activity by Sae2/CtIP

In Saccharomyces cerevisiae (S.cerevisiae), Mre11-Rad50-Xrs2 (MRX)-Sae2 nuclease activity is required for the resection of DNA breaks with secondary structures or protein blocks, while in humans, the MRE11-RAD50-NBS1 (MRN) homolog with CtIP is needed to initiate DNA end resection of all breaks. Phosphorylated Sae2/CtIP stimulates the endonuclease activity of MRX/N. Structural insights into the activation of the Mre11 nuclease are available only for organisms lacking Sae2/CtIP, so little is known about how Sae2/CtIP activates the nuclease ensemble. Here, we uncover the mechanism of Mre11 activation by Sae2 using a combination of AlphaFold2 structural modeling of biochemical and genetic assays. We show that Sae2 stabilizes the Mre11 nuclease in a conformation poised to cleave substrate DNA. Several designs of compensatory mutations establish how Sae2 activates MRX invitro and invivo, supporting the structural model. Finally, our study uncovers how human CtIP, despite considerable sequence divergence, employs a similar mechanism to activate MRN.

Xrs2/NBS1 promote end-bridging activity of the MRE11-RAD50 complex

DNA double strand breaks (DSBs) can be detrimental to the cell and need to be efficiently repaired. A first step in DSB repair is to bring the free ends in close proximity to enable ligation by non-homologous end-joining (NHEJ), while the more precise, but less available, repair by homologous recombination (HR) requires close proximity of a sister chromatid. The human MRE11-RAD50-NBS1 (MRN) complex, Mre11-Rad50-Xrs2 (MRX) in yeast, is involved in both repair pathways. Here we use nanofluidic channels to study, on the single DNA molecule level, how MRN, MRX and their constituents interact with long DNA and promote DNA bridging. Nanofluidics is a suitable method to study reactions on DNA ends since no anchoring of the DNA end(s) is required. We demonstrate that NBS1 and Xrs2 play important, but differing, roles in the DNA tethering by MRN and MRX. NBS1 promotes DNA bridging by MRN consistent with tethering of a repair template. MRX shows a “synapsis-like” DNA end-bridging, stimulated by the Xrs2 subunit. Our results highlight the different ways MRN and MRX bridge DNA, and the results are in agreement with their key roles in HR and NHEJ, respectively, and contribute to the understanding of the roles of NBS1 and Xrs2 in DSB repair.

Sequence and chromatin features guide DNA double-strand break resection initiation

DNA double-strand breaks (DSBs) are cytotoxic genome lesions that must be accurately and efficiently repaired to ensure genome integrity. In yeast, the Mre11-Rad50-Xrs2 (MRX) complex nicks 5'-terminated DSB ends to initiate nucleolytic processing of DSBs for repair by homologous recombination. How MRX-DNA interactions support 5' strand-specific nicking and how nicking is influenced by the chromatin context have remained elusive. Using a deep sequencing-based assay, we mapped MRX nicks at single-nucleotide resolution next to multiple DSBs in the yeast genome. We observed that the DNA end-binding Ku70-Ku80 complex directed DSB-proximal nicks and that repetitive MRX cleavage extended the length of resection tracts. We identified a sequence motif and a DNA meltability profile that is preferentially nicked by MRX. Furthermore, we found that nucleosomes as well as transcription impeded MRX incisions. Our findings suggest that local DNA sequence and chromatin features shape the activity of this central DSB repair complex.

The Ku complex promotes DNA end-bridging and this function is antagonized by Tel1/ATM kinase.

DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ). NHEJ is induced by the binding to DSBs of the Ku70-Ku80 heterodimer, which acts as a hub for the recruitment of downstream NHEJ components. An important issue in DSB repair is the maintenance of the DSB ends in close proximity, a function that in yeast involves the MRX complex and Sae2. Here, we provide evidence that Ku contributes to keep the DNA ends tethered to each other. The ku70-C85Y mutation, which increases Ku affinity for DNA and its persistence very close to the DSB ends, enhances DSB end-tethering and suppresses the end-tethering defect of sae2Δ cells. Impairing histone removal around DSBs either by eliminating Tel1 kinase activity or nucleosome remodelers enhances Ku persistence at DSBs and DSB bridging, suggesting that Tel1 antagonizes the Ku function in supporting end-tethering by promoting nucleosome removal and possibly Ku sliding inwards. As Ku provides a block to DSB resection, this Tel1 function can be important to regulate the mode by which DSBs are repaired.

Development of New Assays for the Simultaneous Measurement of DNA Double‐Strand Break Repair by Multiple Pathways

Cellular DNA is damaged by both endogenous and exogenous sources. Among the different types of DNA lesions that are formed, double‐strand breaks (DSBs) are considered to be the most consequential lesions. They can cause genomic instability and cellular death if not repaired via one of two pathways: nonhomologous end‐joining (NHEJ) or homologous recombination (HR). Deficiency of DSB repair is implicated in several congenital diseases and cancer‐prone disorders. In the budding yeast Saccharomyces cerevisiae NHEJ is accomplished by the actions of the Yku, Mrx, and Dnl4 protein complexes. Recent experiments in this lab have demonstrated that, in contrast to Yku and Dnl4, the Mrx complex is required for NHEJ repair of some DSB end structures but not others. The major goal of the current project is to characterize the role of Mrx and other protein complexes in repair by NHEJ and HR using newly developed plasmid and PCR fragment‐based assays. Experiments performed for the project demonstrated that repair of DSBs with 3′ single‐stranded DNA overhangs was reduced in yku70, mre11 and dnl4 mutants; however, the defect in NHEJ was strongly suppressed when EXO1, encoding a major 5′‐to‐3′ exonuclease, was inactivated in mre11 cells, i.e., in mre11 exo1 double mutants. By contrast, absence of the Sgs1‐Dna2 nuclease‐helicase complex (in mre11 sgs1 mutants) had no effect. NHEJ repair of DSBs with 5′ single‐stranded DNA overhangs was not changed by inactivation of EXO1 or SGS1. These findings implicate Mrx as a potential protector of DSB ends with 3′ overhangs during NHEJ repair and Exo1 as the main degradative nuclease. Additionally, we have developed new plasmid‐ and PCR fragment‐based repair assays that can analyze NHEJ and HR simultaneously using auxotrophic markers and type IIS restriction endonucleases. We anticipate that the new methods for simultaneous measurement will permit exploration of pathway choice in ways not previously possible.

Nej1 interacts with Sae2 at DNA double-stranded breaks to inhibit DNA resection

The two major pathways of DNA double-strand break repair, nonhomologous end-joining and homologous recombination, are highly conserved from yeast to mammals. The regulation of 5′-DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends, and more recently, it was shown to participate in DNA end-bridging and in the inhibition of 5′-resection mediated by the nuclease/helicase complex Dna2–Sgs1. Here, we show that Nej1 interacts with Sae2 to impact DSB repair in three ways. First, we show that Nej1 inhibits interaction of Sae2 with the Mre11–Rad50–Xrs2 complex and Sae2 localization to DSBs. Second, we found that Nej1 inhibits Sae2-dependent recruitment of Dna2 independently of Sgs1. Third, we determined that NEJ1 and SAE2 showed an epistatic relationship for end-bridging, an event that restrains broken DNA ends and reduces the frequency of genomic deletions from developing at the break site. Finally, we demonstrate that deletion of NEJ1 suppressed the synthetic lethality of sae2Δ sgs1Δ mutants, and that triple mutant viability was dependent on Dna2 nuclease activity. Taken together, these findings provide mechanistic insight to how Nej1 functionality inhibits the initiation of DNA resection, a role that is distinct from its involvement in end-joining repair at DSBs.

Rif2 protects Rap1-depleted telomeres from MRX-mediated degradation in Saccharomyces cerevisiae.

Rap1 is the main protein that binds double-stranded telomeric DNA in Saccharomyces cerevisiae. Examination of the telomere functions of Rap1 is complicated by the fact that it also acts as a transcriptional regulator of hundreds of genes and is encoded by an essential gene. In this study, we disrupt Rap1 telomere association by expressing a mutant telomerase RNA subunit (tlc1-tm) that introduces mutant telomeric repeats. tlc1-tm cells grow similar to wild-type cells, although depletion of Rap1 at telomeres causes defects in telomere length regulation and telomere capping. Rif2 is a protein normally recruited to telomeres by Rap1, but we show that Rif2 can still associate with Rap1-depleted tlc1-tm telomeres, and that this association is required to inhibit telomere degradation by the MRX complex. Rif2 and the Ku complex work in parallel to prevent tlc1-tm telomere degradation; tlc1-tm cells lacking Rif2 and the Ku complex are inviable. The partially redundant mechanisms may explain the rapid evolution of telomere components in budding yeast species.

Sir3 heterochromatin protein promotes non-homologous end joining by direct inhibition of Sae2.

Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in gene expression regulation and maintenance of genome stability. How heterochromatin proteins regulate DNA repair remains poorly described. In the yeast Saccharomyces cerevisiae, the silent information regulator (SIR) complex assembles heterochromatin-like chromatin at sub-telomeric chromosomal regions. SIR-mediated repressive chromatin limits DNA double-strand break (DSB) resection, thus protecting damaged chromosome ends during homologous recombination (HR). As resection initiation represents the crossroads between repair by non-homologous end joining (NHEJ) or HR, we asked whether SIR-mediated heterochromatin regulates NHEJ. We show that SIRs promote NHEJ through two pathways, one depending on repressive chromatin assembly, and the other relying on Sir3 in a manner that is independent of its heterochromatin-promoting function. Via physical interaction with the Sae2 protein, Sir3 impairs Sae2-dependent functions of the MRX (Mre11-Rad50-Xrs2) complex, thereby limiting Mre11-mediated resection, delaying MRX removal from DSB ends, and promoting NHEJ.

Antagonistic relationship of NuA4 with the non-homologous end-joining machinery at DNA damage sites.

The NuA4 histone acetyltransferase complex, apart from its known role in gene regulation, has also been directly implicated in the repair of DNA double-strand breaks (DSBs), favoring homologous recombination (HR) in S/G2 during the cell cycle. Here, we investigate the antagonistic relationship of NuA4 with non-homologous end joining (NHEJ) factors. We show that budding yeast Rad9, the 53BP1 ortholog, can inhibit NuA4 acetyltransferase activity when bound to chromatin in vitro. While we previously reported that NuA4 is recruited at DSBs during the S/G2 phase, we can also detect its recruitment in G1 when genes for Rad9 and NHEJ factors Yku80 and Nej1 are mutated. This is accompanied with the binding of single-strand DNA binding protein RPA and Rad52, indicating DNA end resection in G1 as well as recruitment of the HR machinery. This NuA4 recruitment to DSBs in G1 depends on Mre11-Rad50-Xrs2 (MRX) and Lcd1/Ddc2 and is linked to the hyper-resection phenotype of NHEJ mutants. It also implicates NuA4 in the resection-based single-strand annealing (SSA) repair pathway along Rad52. Interestingly, we identified two novel non-histone acetylation targets of NuA4, Nej1 and Yku80. Acetyl-mimicking mutant of Nej1 inhibits repair of DNA breaks by NHEJ, decreases its interaction with other core NHEJ factors such as Yku80 and Lif1 and favors end resection. Altogether, these results establish a strong reciprocal antagonistic regulatory function of NuA4 and NHEJ factors in repair pathway choice and suggests a role of NuA4 in alternative repair mechanisms in situations where some DNA-end resection can occur in G1.

The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks.

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5'-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.

DNA polymerases η and κ bypass N2-guanine-O6-alkylguanine DNA alkyltransferase cross-linked DNA-peptides

DNA-protein cross-links are formed when proteins become covalently trapped with DNA in the presence of exogenous or endogenous alkylating agents. If left unrepaired, they inhibit transcription as well as DNA unwinding during replication and may result in genome instability or even cell death. The DNA repair protein O6-alkylguanine DNA-alkyltransferase (AGT) is known to form DNA cross-links in the presence of the carcinogen 1,2-dibromoethane, resulting in G:C to T:A transversions and other mutations in both bacterial and mammalian cells. We hypothesized that AGT-DNA cross-links would be processed by nuclear proteases to yield peptides small enough to be bypassed by translesion (TLS) polymerases. Here, a 15-mer and a 36-mer peptide from the active site of AGT were cross-linked to the N2 position of guanine via conjugate addition of a thiol containing a peptide dehydroalanine moiety. Bypass studies with DNA polymerases (pols) η and κ indicated that both can accurately bypass the cross-linked DNA peptides. The specificity constant (kcat/Km) for steady-state incorporation of the correct nucleotide dCTP increased by 6-fold with human (h) pol κ and 3-fold with hpol η, with hpol η preferentially inserting nucleotides in the order dC > dG > dA > dT. LC-MS/MS analysis of the extension product also revealed error-free bypass of the cross-linked 15-mer peptide by hpol η. We conclude that a bulky 15-mer AGT peptide cross-linked to the N2 position of guanine can retard polymerization, but that overall fidelity is not compromised because only correct bases are inserted and extended.

Local nucleosome dynamics and eviction following a double-strand break are reversible by NHEJ-mediated repair in the absence of DNA replication

We interrogated at nucleotide resolution the spatiotemporal order of chromatin changes that occur immediately following a site-specific double-strand break (DSB) upstream of the PHO5 locus and its subsequent repair by nonhomologous end joining (NHEJ). We observed the immediate eviction of a nucleosome flanking the break and the repositioning of adjacent nucleosomes away from the break. These early chromatin events were independent of the end-processing Mre11-Rad50-Xrs2 (MRX) complex and preceded the MRX-dependent broad eviction of histones and DNA end-resectioning that extends up to ∼8 kb away from the break. We also examined the temporal dynamics of NHEJ-mediated repair in a G1-arrested population. Concomitant with DSB repair by NHEJ, we observed the redeposition and precise repositioning of nucleosomes at their originally occupied positions. This re-establishment of the prelesion chromatin landscape suggests that a DNA replication-independent mechanism exists to preserve epigenome organization following DSB repair.

Sae2 and Rif2 regulate MRX endonuclease activity at DNA double-strand breaks in opposite manners

SummaryThe Mre11-Rad50-Xrs2 (MRX) complex detects and processes DNA double-strand breaks (DSBs). Its DNA binding and processing activities are regulated by transitions between an ATP-bound state and a post-hydrolysis cutting state that is nucleolytically active. Mre11 endonuclease activity is stimulated by Sae2, whose lack increases MRX persistence at DSBs and checkpoint activation. Here we show that the Rif2 protein inhibits Mre11 endonuclease activity and is responsible for the increased MRX retention at DSBs in sae2Δ cells. We identify a Rad50 residue that is important for Rad50-Rif2 interaction and Rif2 inhibition of Mre11 nuclease. This residue is located near a Rad50 surface that binds Sae2 and is important in stabilizing the Mre11-Rad50 (MR) interaction in the cutting state. We propose that Sae2 stimulates Mre11 endonuclease activity by stabilizing a post-hydrolysis MR conformation that is competent for DNA cleavage, whereas Rif2 antagonizes this Sae2 function and stabilizes an endonuclease inactive MR conformation.

Single Molecule Experiments Define the Role of Nbs1 and Xrs2 in DNA Tethering by MRN and MRX in DSB Repair

To ensure genome stability and hinder introduction of disease-causing changes, DNA double-strand breaks (DSB) need to be repaired. Homologous recombination (HR) is a key DSB repair pathway. At the start of HR, the MRE11-RAD50-NBS1 (MRN) complex finds the broken ends and upon stimulation of its nuclease activity by CtIP performs the DSB processing for repair. The repair process involves DNA tethering, which is poorly understood. in this study, we use single-molecule analysis to investigate how MRN interacts with long DNA. The method is based on confining long DNA molecules in nanochannels. in contrast to tethering one or both ends to a substrate, as in most common single DNA molecule methods, the DNA is completely free in the nano channels, meaning that reaction on the DNA ends can be studied. Single molecule experiments with MRN and DNA in nanochannels show direct evidence of a strong DNA interaction and tethering of distant DNA fragments by MRN. When comparing MRN and the yeast homologue Mre11-Rad50-Xrs2 (MRX), we observed key similarities. in both systems, the absence of NBS1/Xrs2, lowers the concatemer and circle formation dramatically. Data from single molecule experiments and EMSA show that NBS1 and Xrs2 alone have a strong DNA interaction. These findings strengthen the theory of Nbs1/Xrs2 playing a role in the localization of the MRN/X complex to DNA ends and mediate DNA annealing. Finally, the addition of the MRN/X co-factor CtIP and Sae2, respectively, shows little to no effect on the bridging by MRN/X, although both CtIP and Sae2 exhibit strong DNA bridging on their own. Thus the interaction between MRN/X and the co-factor that is crucial for the nuclease activity is potentially not important for DNA bridging.

A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.

A Rad51-independent pathway promotes single-strand template repair in gene editing.

The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.

The 9-1-1 Complex Controls Mre11 Nuclease and Checkpoint Activation during Short-Range Resection of DNA Double-Strand Breaks

Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process in which an initial short-range step is catalyzed by the Mre11-Rad50-Xrs2 (MRX) complex and limited to the vicinity of the DSB end. Then the two long-range resection Exo1 and Dna2-Sgs1 nucleases extend the resected DNA tracts. How short-range resection is regulated and contributes to checkpoint activation remains to be determined. Here, we show that abrogation of long-range resection induces a checkpoint response that decreases DNA damage resistance. This checkpoint depends on the 9-1-1 complex, which recruits Dpb11 and Rad9 at damaged DNA. Furthermore, the 9-1-1 complex, independently of Dpb11 and Rad9, restricts short-range resection by negatively regulating Mre11 nuclease. We propose that 9-1-1, which is loaded at the leading edge of resection, plays a key function in regulating Mre11 nuclease and checkpoint activation once DSB resection is initiated.

DNA binding modes influence Rap1 activity in the regulation of telomere length and MRX functions at DNA ends.

The cellular response to DNA double-strand breaks (DSBs) is initiated by the Mre11–Rad50–Xrs2 (MRX) complex that has structural and catalytic functions. MRX association at DSBs is counteracted by Rif2, which is known to interact with Rap1 that binds telomeric DNA through two tandem Myb-like domains. Whether and how Rap1 acts at DSBs is unknown. Here we show that Rif2 inhibits MRX association to DSBs in a manner dependent on Rap1, which binds to DSBs and promotes Rif2 association to them. Rap1 in turn can negatively regulate MRX function at DNA ends also independently of Rif2. In fact, a characterization of Rap1 mutant variants shows that Rap1 binding to DNA through both Myb-like domains results in formation of Rap1-DNA complexes that control MRX functions at both DSBs and telomeres primarily through Rif2. By contrast, Rap1 binding to DNA through a single Myb-like domain results in formation of high stoichiometry complexes that act at DNA ends mostly in a Rif2-independent manner. Altogether these findings indicate that the DNA binding modes of Rap1 influence its functional properties, thus highlighting the structural plasticity of this protein.

Tel1 Activation by the MRX Complex Is Sufficient for Telomere Length Regulation but Not for the DNA Damage Response in Saccharomyces cerevisiae.

Previous models suggested that regulation of telomere length in Saccharomyces cerevisiae by Tel1(ATM) and Mec1(ATR) would parallel the established pathways regulating the DNA damage response. Here, we provide evidence that telomere length regulation differs from the DNA damage response in both the Tel1 and Mec1 pathways. We found that Rad53 mediates a Mec1 telomere length regulation pathway but is dispensable for Tel1 telomere length regulation, whereas in the DNA damage response, Rad53 is regulated by both Mec1 and Tel1 Using epistasis analysis with a Tel1 hypermorphic allele, Tel1-hy909, we found that the MRX complex is not required downstream of Tel1 for telomere elongation but is required downstream of Tel1 for the DNA damage response. Our data suggest that nucleolytic telomere end processing is not a required step for telomerase to elongate telomeres.