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.
Replication is a major source of nuclear DNA damage under normal mitotic growth conditions, i. e. in the absence of drugs or irradiation [1]. Studies in both prokaryotes and eukaryotes suggest that replication fork movement rates are heterogeneous, with slower fork movement, fork pausing and breaks at “hard-to-replicate” regions of the genome [2]. Stalled or broken replication forks are recognized as sites of DNA damage and activate a DDR (DNA damage response). The recognition of DNA damage leads to activation of a signaling cascade: phosphorylation is used to transduce the signal from the sensor kinases ATR and ATM (Mec1 and Tel1, respectively, in budding yeast) to the adaptors (Rad9 and Mrc1 in yeast), and then to the effector kinases Chk1 and Chk2 (Chk1 and Rad53/Dun1 in yeast), which in turn induce cell cycle arrest and DNA repair [3]. Broken replication forks generate a one-ended DSB that can be repaired by different mechanisms relying on DNA homology between the broken end and unbroken sister chromatid: homologous recombination, synthesis dependent strand annealing, or break-induced replication (BIR). During BIR, DSB processing by exo- and endonucleases generates a 3′ssDNA overhang that enables the homologous recombination machinery (Rad51/52/54/55/57) to invade a homologous sequence of the unbroken sister chromatid to re-establish the DNA synthesis [4]. Unlike conventional DNA replication, BIR employs a conservative replication mode [5], [6] and requires the Polδ subunit Pol32 and the 5′→3′ helicase Pif1 [7], [8]. Replication of telomeres, the ends of eukaryotic chromosomes, involves the enzyme telomerase. The core telomerase complex of S. cerevisiae includes an RNA component TLC1 [9] and the reverse transcriptase Est2 [10], [11]. Telomerase-dependent telomere synthesis is tightly coupled to conventional DNA replication and occurs in the S/G2 phase of the cell cycle [12], [13]. Telomerase can also add a telomere to a non-telomeric DNA end [14], [15], such as a DSB or a broken replication fork, thereby leading to a terminal deletion [16]. To prevent de novo telomere addition, the Pif1 helicase is phosphorylated in cells with DNA damage and the phosphorylated form of the protein inhibits telomerase at broken DNA ends [17]. Pif1 helicase is also a negative regulator of telomerase at telomeres under normal growth conditions [16]. Therefore, cells lacking Pif1 possess both longer telomeres and elevated frequencies of de novo telomere addition to DSBs [16], [18]. Although telomerase is inhibited at broken DNA ends through the phosphorylation of Pif1 by the DNA damage signaling machinery, as yet it remains unknown if any modulation of telomere synthesis in response to DNA damage takes place. Here we report that the DNA damage-induced phosphorylation of Pif1 is required not only for the inhibition of telomerase at DSBs but also for the function of Pif1 in BIR. In turn, activation of BIR in cells with DNA damage leads to telomere lengthening which may provide an additional layer of telomere protection against the DNA repair machinery [19]. Thus, the DNA damage-induced phosphorylation of Pif1 promotes potentially telomere-stabilizing telomere extension while repressing deleterious de novo telomere addition events at DNA breaks, thereby funneling the broken DNA into appropriate genome-preserving repair pathways.
During DNA replication, incomplete DNA ligation via partially abrogated ligase function may cause DNA discontinuities. In S. cerevisiae, CDC9 encodes a replicative DNA ligase essential for yeast cell viability [20]. Like other temperature sensitive cdc9 mutants, cdc9-1 cells grow normally at 23°C but at 36°C arrest as large-budded cells with the nuclei at the bud necks [21]. The cdc9-1 arrest is temporarily relieved in cdc9-1 rad9Δ cells, which at the non-permissive temperature undergo one or two cell divisions before losing viability [22]. It has been reported previously that cdc9-1 mutants have longer telomeres [23]. When grown at 22°C, the cdc9-1 mutant strain possessed a bulk telomere length similar to CDC9 cells (Figure 1A). However, when cdc9-1 yeast were propagated at an intermediate temperature, 26°C, after about 80 generations the telomeres were distinctly and reproducibly elongated (mean telomere length 450 bp) compared to the isogenic CDC9 strain (mean telomere length 350 bp) (Figure 1A). At this semi-permissive temperature (26°C), cdc9-1 cells showed only a very mild growth defect phenotype and possessed a slightly smaller colony size (Figure 1B). The telomere length phenotype in cdc9-1 cells could have arisen from any of the following possibilities: (i) increased telomerase action, (ii) increased telomeric recombination, (iii) impaired telomere processing/shortening. To distinguish between these possibilities, we compared the dynamics of telomere length changes in response to cdc9-1 in the presence and absence of telomerase. The double heterozygous diploids CDC9/cdc9-1 EST2/est2Δ and CDC9/cdc9-1 TLC1/tlc1Δ were sporulated and germinated spores grown into single colonies (about 20 generations) at 22°C. The four progeny spores from the tetrads were subsequently grown on plates at 26°C for a further ∼40 generations (two streaks) for telomere length analysis. As expected, in the presence of telomerase, the cdc9-1 cells possessed longer telomeres than CDC9 cells (Figure 1C). However, in cells lacking Est2 or TLC1, the cdc9-1 mutation had no effect on telomere length: both CDC9 and cdc9-1 cells lacking either essential telomerase component showed similar rates of telomere shortening. Thus, the telomere lengthening in response to cdc9-1 was dependent on functional telomerase and was not a result of impaired telomere erosion or end processing. FACS analysis indicated that non-synchronized populations of haploid cdc9-1 yeast were enriched for cells with 2n DNA content (Figure 2A), suggestive of a cell cycle progression delay in late S-phase or G2, perhaps due to checkpoint activation. In response to DNA damage, a central component of the DNA damage signaling pathway, Rad53, is activated by phosphorylation [24]. In cdc9-1 cells at 26°C, but not 22°C, a shift in Rad53 mobility upon SDS-PAGE characteristic of Rad53 phosphorylation in response to DNA damage was observed (Figure 2B). Therefore, at the semi-permissive temperature the cdc9-1 mutation leads to DDR activation. We tested the genetic dependency of the telomere lengthening in cdc9-1 cells at 26°C on known DNA damage signaling network components. Diploid strains heterozygous for both CDC9/cdc9-1 and each gene of interest were sporulated and the spores germinated at 22°C. In the case of MEC1 or RAD53, the cells were also heterozygous for SML1/sml1Δ in order to suppress the known lethality of mec1Δ or rad53Δ with sml1Δ in the haploid progeny [25]. Progeny with different genotypes were propagated at 26°C for 4 re-streaks (∼80 generations) to allow telomere length to reach the stable length characteristic of each genotype. Telomere elongation in cdc9-1 cells was retained in a tel1Δ or chk1Δ background, in the replication checkpoint deficient mutant mrc1-AQ [26] and in an sml1Δ dun1Δ background where sml1Δ suppresses the effect of dun1Δ on S-phase progression [27]. In contrast, the telomere lengthening in cdc9-1 cells was dependent on RAD50, MEC3, RAD24 (to some extent), MEC1, RAD9, and RAD53 (Figure 2C). Cdc17 is a catalytic subunit of the DNA polymerase Polα that plays a pivotal role in the coordination of telomere synthesis and conventional DNA replication [28]. Like cdc9-1, a temperature sensitive mutation, cdc17-1, results in longer telomeres [23], [29]. However, cdc17-1 did not exhibit constitutive activation of DDR and the cdc17-1 induced telomere lengthening was independent of the DNA damage checkpoint (Figure S1). Therefore, we conclude that the telomere elongation phenotype of cdc9-1 at 26°C is not a defect related to the coordination of lagging strand synthesis at telomeres, but rather depends on activation of the central DNA damage checkpoint network (RAD50, MEC3, RAD24, MEC1, RAD9, and RAD53) but not on TEL1, MRC1, DUN1 or CHK1. Thus, the effect of cdc9-1 on telomere synthesis is likely to be indirect, via activation of the DNA damage signaling which then leads to telomere lengthening. We used a candidate approach to identify a potential link between the activation of the DNA damage response and telomere lengthening in cdc9-1 cells based on (i) a previously reported role for the candidate proteins in telomere metabolism; (ii) the candidate proteins are regulated by the DNA damage signaling machinery. A set of candidates came from the report that activation of DDR leads to dynamic changes in telomere architecture [30]. The yeast telomeric chromatin components Rap1, Sir2/3/4, Rif1, and Rif2 (which regulate telomerase action on a telomere in cis [31]), and the Ku70/80 complex (which is required for telomerase localization in the nucleus and at the telomeres [32]) re-localize away from telomeres in a Rad9-dependent manner [30]. Such re-localization during DDR activation in cdc9-1 could lead to longer telomeres. We analyzed the effect of the cdc9-1 mutation on telomere length in cells deleted for RIF1, RIF2, SIR2, SIR3, SIR4, YKU70, or YKU80. The telomere length increase observed in cdc9-1 cells did not depend on RIF1 or RIF2 but was moderately decreased in the absence of the Sir proteins, and no telomere elongation was observed in either yku70 or yku80 backgrounds (Figure S2). Therefore, the telomere elongation observed in cdc9-1 cells is dependent on Ku70/80 and at least partially dependent on Sir proteins. The Ku70/80 dependence is in accord with the known requirement of Ku70/80 in telomerase recruitment [32], while the SIR-dependence could reflect known roles in telomere architecture and telomerase regulation in cis, or as yet unexplored role (s) in BIR-dependent telomere replication. DNA damage-induced signaling is also known to regulate a nuclear form of the Pif1 helicase [17], a well characterized inhibitor of telomerase at both telomeres and DNA breaks [16]. Phosphorylation of nuclear Pif1 (nPif1) in response to DSBs is required for telomerase inhibition at broken DNA ends [17]. To query if nPif1 and its phosphorylation during the DDR were responsible for the telomere lengthening in cdc9-1 cells, we combined cdc9-1 with pif1Δ, pif1-m2 (nPif1 null [16]), or the previously reported PIF1 alleles pif1-3A and pif1-4A that abrogate Mec1-Rad53-dependent phosphorylation of the peptide TLSSAES [17]. Like cdc9-1, pif1Δ and pif1-m2 have longer telomeres but no additive telomere lengthening was observed in either cdc9-1 pif1Δ or cdc9-1 pif1-m2 (Figure 3A). Furthermore, both pif1-3A and pif1-4A mutations alleviated the cdc9-1 induced telomere lengthening, with slightly more pronounced effect in a pif1-4A background compared with pif1-3A (Figure 3B), suggesting a possibility of nPif1 phosphorylation playing a role in telomere elongation in cdc9-1 cells. Thus, the telomere lengthening in cdc9-1 cells is genetically dependent on the presence of the nuclear form of Pif1 and specifically on the phosphorylation of TLSSAES. To test whether nPif1 was phosphorylated in cdc9-1 cells, we immunoprecipitated Pif1-4myc from CDC9 and cdc9-1 cells and treated a third of each sample with either CIP or λ phosphatase and compared their mobility by SDS-PAGE (Figure 4A). Indeed, nPif1 migration was retarded to a greater extent in cdc9-1 than in CDC9 cells and phosphatase treatment eliminated this mobility difference. In fact, phosphatase treatment resulted in faster mobility nPif1 species in both CDC9 and cdc9-1 cells (Figure 4A: compare lane 2 to lanes 4 and 6). Together, these findings suggest that nPif1 possesses a basal level of phosphorylation in wild-type cells, and that additional phosphorylation events occur in response to cdc9-1. Consistent with the DDR-dependent modulation of nPif1 activity at DSBs, nPif1 phosphorylation in response to cdc9-1 depended on MEC1 and RAD53 but not TEL1 or DUN1 (Figure 4B). To further probe nPif1phosphorylation, we used an antibody specific to the nPif1 phospho-regulatory locus (pT) LS (pS) AE (anti-P-Pif1 antibody) [17] and established its phosphatase-dependent recognition of nPif1 in cdc9-1 (but not CDC9) cells that was largely abrogated by the pif1-4A mutation (Figure 4C, top panel). In sml1Δ mec1Δ and sml1Δ rad53Δ backgrounds, the cdc9-1 dependent recognition of Pif1 with the anti-P-Pif1 antibody was considerably reduced (Figure 4D). Taken together, the data indicate that cdc9-1 activates MEC1-RAD53-dependent phosphorylation of nPif1 on TLSSAES (as well as on other positions on Pif1), and that the resulting telomere lengthening minimally requires TLSSAES phosphorylation. In addition to its roles in regulation of telomerase at telomeres and DSBs [16], [17], nPif1 also has been reported to have a role in BIR [8]. The cdc9-1 mutation causes replicative ligase insufficiency which results in nicks as evidenced from accumulation of unligated nascent DNA strands [20], [33]. Passage of replication forks through these nicks would lead to broken replication forks [34] that would require homology-mediated repair, such as homologous recombination or BIR. To test if BIR had any role in telomere lengthening in cdc9-1 cells, we combined cdc9-1 with deletions in genes encoding the major components of BIR machinery, such as Rad51, Rad52, and Pol32. Indeed, we observed that RAD51, RAD52, and POL32 were required for the cdc9-1 induced telomere elongation (Figure 5A). Further, the cdc9-1 induced accumulation of G2 cells was retained in pif1-4A and pol32Δ backgrounds (Figure S3) despite the absence of telomere lengthening (see Figures 3B and 5A), suggesting that PIF1 and POL32 are downstream of the DDR activation in the pathway leading to telomere elongation in cdc9-1 cells. Because Pol32 and nPif1 are required specifically for BIR [7], [8] but not for homologous recombination, our data suggest that BIR plays a major role in telomere elongation in cdc9-1 yeast. To explore this hypothesis further we tested if the PCSS complex (Psy3/Csm2/Shu1/Shu2) and Mph1 helicase, known to regulate Rad51 filament formation and post-invasion steps in BIR respectively [35], [36], [37], had an effect on telomere length in cdc9-1. Deletion of PSY3 partially suppressed telomere elongation in cdc9-1 cells (Figure 5B), consistent with the previously reported role for the PCSS complex as a positive regulator of Rad51 filament assembly on a processed DSB [35], [36]. Mph1 disrupts D-loops formed upon invasion of Rad51-covered ssDNA into a homologous sequence [37] and MPH1 overexpression from the GAL1 promoter inhibits BIR [38]. Overexpression of MPH1 in cdc9-1 cells resulted in alleviation of telomere elongation (Figure 5C) consistent with the hypothesis that telomere elongation is BIR-dependent. Thus, the telomere elongation in cdc9-1 is genetically dependent on core components of BIR machinery Rad51, Rad52, nPif1, Pol32 as well as on its regulators Mph1 and the PCSS complex. Since telomere elongation in response to cdc9-1 was dependent on both BIR and nPif1 phosphorylation on TLSSAES, we tested if the nPif1 TLSSAES phosphorylation was required for BIR using a well-established system of galactose-inducible HO endonuclease expression to generate a DSB [39] next to two selectable markers, each located on either side of the break (see Figure 6A for details). The KAN selectable marker on the centromere-proximal side of the break was further separated into two non-functional fragments on CHR VIIL and CHR IIR, such that a functional allele of KAN would be restored only when BIR initiation at CHR IIR provided the template for repair of DSB on CHR VIIL (Figure 6A). Consistent with previously reported data [8], PIF1 cells were more efficient at BIR than pif1-m2, and the pif1-4A mutants exhibited a loss of function similar to pif1-m2 (Figure 6B). Both pol32 and rad9 abrogated PIF1-dependent BIR (Figure 6B), suggesting that nPif1 requires functional Polδ and DDR to promote BIR. Since de novo telomere addition is upregulated in pif1-4A cells [17] and thus might compete with BIR for DSBs, we tested whether est2 or yku80 mutations, which disrupt de novo telomere addition [18], affected BIR in PIF1 and pif1-4A cells and found that the increased de novo telomere addition in pif1-4A did not impede BIR (Figure 6B). Therefore, the phosphorylation of nPif1 at the TLSSAES locus is required specifically for its role in BIR that is not affected by de novo telomere addition events that occur at DSBs. Our hypothesis that cdc9-1 cells suffer from broken replication forks as a result of replication passing through nicks is based on (i) the known role of Cdc9 in replication as a ligase of newly synthesized DNA strands [20], (ii) observed accumulation of unligated nascent DNA strands in the cdc9-1 mutant [20], [33] which suggests nicks or single-stranded gaps, (iii) the established fact that replication through a nick results in a broken replication fork [34], and (iv) RAD9-dependent cell cycle arrest of cdc9-1 mutants at the non-permissive temperature [20], [21] which can be explained by the existence of broken replication forks. If the hypothesis that broken replication forks repaired by BIR contribute to telomere elongation is correct, then there should be mutations in other components of replication machinery that also result in longer telomeres due to increased breakage of replication forks and activation of a DDR and BIR. Rrm3 is a non-essential DNA helicase required for replication through “hard-to-replicate” regions and the loss of RRM3 results in a higher rate of fork breakage, constitutive activation of a DDR, and slight telomere elongation that is PIF1-dependent [40], [41]. cdc44-5 is a temperature-sensitive mutant allele of Replication Factor C which operates as a PCNA clamp loader. At 30°C, cdc44-5 mutants possess longer telomeres and accumulate cells in G2 [23]. We analyzed both rrm3Δ and cdc44-5 for constitutive activation of a DDR and telomere elongation, and the possible dependence of the latter on Pol32 and nPif1. The DDR was constitutively activated in both rrm3Δ and cdc44-5, with more pronounced Rad53 phosphorylation observed in cdc44-5 cells (Figure S4A). The slight telomere elongation in rrm3Δ was abolished by the pif1-4A mutation and there was no further increase in telomere length in rrm3Δ pol32Δ cells compared to either single mutant alone (Figure S4B). Combining cdc44-5 with pol32Δ resulted in a synthetic lethality (Figure S4C) suggesting that BIR might be constitutively active in cdc44-5 and required for cell viability (or that the combination of the two mutations could be too disruptive for conventional replication). The extensive telomere lengthening in cdc44-5 cells was strongly suppressed by the pif1-4A mutation (Figure S4D). Therefore, both rrm3Δ and cdc44-5 resemble cdc9-1 in their constitutive activation of DDR and longer telomere phenotype and, like cdc9-1, telomere elongation in rrm3Δ cells is dependent on the BIR components nPif1 and Pol32 and the long telomere phenotype in cdc44-5 is dependent on nPif1.
The DDR-dependence of telomere elongation in cdc9-1 cells differed from the telomere elongation observed in cdc17-1 cells, which is not DDR-dependent (Figure S1). We suggest that the effect of cdc9-1 on telomere length is indirect, i. e. it is not due to uncoordinated chromosome replication and telomerase-dependent telomere lengthening. Instead, the DDR in cdc9-1 cells is required for successful repair of broken replication forks via BIR that in turn causes additional telomere lengthening. We propose that this mechanism might explain previous reports that rrm3 mutants, which suffer from replication fork pausing and breakage, also show constitutive activation of Rad53 and longer telomeres [40], [46]. A combination of constitutive damage signaling and telomere lengthening is also seen in cdc44-5 cells. It is unknown whether the telomere lengthening in these mutants is DDR-dependent (rrm3 cells require RAD53 for their viability [46]), but the telomere elongation in both mutants was dependent on the DNA damage induced phosphorylation of nPif1. Pol32 was also required for longer telomeres in rrm3 cells and the synthetic lethality between cdc44-5 and pol32Δ suggests a possibility that BIR is critical for cdc44-5 cell viability. In the genome-wide screens for S. cerevisiae deletion mutants causing telomere lengthening or shortening phenotypes, many genes that are relevant to DNA metabolism through replication, repair or chromatin structure have been reported [47], [48]. We suggest that like cdc9-1, the telomere length effects of some of these mutations may result from constitutive generation of DNA damage. The dependency of the cdc9-1 induced telomere lengthening on the DNA damage signaling is likely to be more complex than the requirement of the signaling kinases for nPif1 phosphorylation. The checkpoint activation is also necessary for the cell cycle arrest to ensure the completion of BIR [43], and therefore is also required for BIR fork passage through telomeres before the cells can enter mitosis. The signaling is also required for re-localization of the telomere bound proteins Sir3 and Ku in response to DNA damage [30]. This event might be relevant to the cdc9-1 induced telomere lengthening as the latter was reduced in the sir mutants and abolished in the yku70 and yku80 backgrounds (Figure S2). The DNA damage induced changes in telomeric chromatin may affect telomerase access to telomeres so that telomerase is recruited to a larger number of telomeres and/or it is allowed longer time to elongate each telomere during S-phase, thereby increasing telomere length in cells with constitutive DNA damage. Our findings suggest that the DDR-induced nPif1 phosphorylation does not only inhibit telomerase at DSBs as previously demonstrated, but it also plays a pivotal role in promoting BIR at broken replication forks and DSBs. Healing a broken DNA end by de novo telomere addition leads to global re-arrangements or partial loss of genetic material, thereby contributing to genome instability. Therefore nPif1 regulation directed at telomerase inhibition at DSBs [17] and facilitation of DSB repair via BIR (see Figure 6B) serve together to ensure genome repair and preservation. At the same time, the increased telomerase activity on telomeres as a result of an active BIR may have additional biological significance. For example, the telomere elongation in response to DNA damage could promote recruitment of additional telomere-associated chromatin factors (Rap1, Rif1, Sir proteins, etc.) that could in turn protect shorter telomeres against the DNA repair machinery activated in response to DNA damage [19]. In addition, the telomere lengthening via BIR could promote repair of critically short telomeres that have previously eroded and activated BIR in sub-telomeric regions. Since BIR leads to telomere lengthening, it may boost telomerase-dependent elongation of critically short telomeres involved in BIR. This mechanism could act in addition to the Tel1-mediated signaling which has been reported to increase telomerase recruitment on a shortened telomere [49], [50]. On the other hand, longer telomeres are harder to replicate as telomeric chromatin causes replication fork stalling [33], [41] and broken replication forks within telomeric repeats would result in truncated telomeres. Truncated telomeres after DSBs or as a result of active trimming [51] could therefore benefit from repair by telomerase activity. Thus, dynamic telomere length adjustments in response to changing extracellular and intracellular environment could have evolved to minimize genomic instability and maximize cell fitness and survival. In summary, we uncovered that telomerase-dependent telomere elongation is modulated in response to DNA damage. Additional elongation occurs as a result of active BIR that requires Mec1-Rad53 dependent phosphorylation of nPif1. Therefore, nPif1 is a multifunctional regulator of telomere synthesis: it inhibits telomerase at telomeres in cells with no DNA damage, whereas in cells with DNA damage, its phosphorylation impairs telomerase action at DSBs and at the same time promotes telomere synthesis via BIR.
For genetic analysis of telomere length in different mutants, teloblot analysis was performed on a minimum of 3 tetrads, often 4-6 tetrads, that originated from at least 2 independently constructed parental strains. Relevant heterozygous diploids of each genotype were sporulated and progeny of at least four tetrads (at least two coming from each of the two diploids) were passaged for ∼80 generations at either 26°C (tetrads containing cdc9-1 mutants) or 30°C (tetrads containing rrm3 or cdc44-5 mutants) on YPD agar (or YPGal agar if induction of GAL1- promoter was required), to equilibrate telomere length. Yeast genomic DNA was purified, digested with KpnI, and resolved on 0. 85% w/v agarose gels. Southern blotting and hybridization were performed as described previously [33]. The random-primer (Prime-It II Kit, Stratagene) radiolabeled probe KL1 (recognizes the telomere proximal 650 bp of Y′ repeats) was used for hybridization in the KpnI experiments. In cases where slight variability in mutant behavior was observed, more than one tetrad (or clone) for the given combination of mutations was included in the corresponding figures (see Figure 3B for cdc9-1 pif1-3A, Figure 5B for cdc9-1 psy3, Figure S1 for cdc17-1 rad53 sml1, and Figure S4 for cdc44-5 pif1-4A). Yeast protein extracts were prepared using TCA precipitation [52]. Proteins were resolved on SDS-PAGE and transferred to PVDF membrane. Immunological detection was performed using ECL+ kit (GE Healthcare) according to the manufacturer' s instructions. Mouse monoclonal α-myc 9E10 (Covance), goat polyclonal α-Rad53 yC-19 (Santa Cruz), and rabbit polyclonal α- VIDFYL (pT) LS (pS) AE (custom made, QCB) antibodies were used for Pif1-4myc, endogenous Rad53, and phosphorylated Pif1 detection respectively. Pif1-4myc was immunoprecipitated from cleared cell lysates at 4°C. Yeast cells were homogenized by bead beating in Lysis Buffer (25 mM HEPES (pH 7. 5), 150 mM KCl, 1 mM EDTA, 1 mM EGTA, 0. 1% v/v NP-40,10% v/v glycerol) with phosphatase inhibitors (50 mM NaF, 50 mM sodium glycerophosphate) and protease inhibitors (Compete, EDTA-free protease inhibitors cocktail tablets, Roche). The lysates were cleared by centrifugation (14K rpm, 20′). Pif1-4Myc was immunoprecipitated by incubation of cleared lysates with 1∶150 dilution of α-myc antibody 9E10 (Covance) for 2 h followed by addition of Protein G agarose beads (Sigma) for an additional 2 h. Beads were washed 3×5′ in the Lysis Buffer and then rinsed in 1× phosphatase buffer supplied with either CIP or λ phosphatase (NEB). Phosphatase treatments were performed at room temperature in the corresponding buffers. Reactions were stopped by addition of SDS-PAGE sample buffer and 5′ boiling. Cells were patched on YPRaffinose plates and grown overnight. Cells were re-suspended in YP broth to OD∼1. Serial dilutions were plated on YPD and YPGalactose agar. In 3 days, YPGalactose plates were replica-plated onto minimal media without uracil and on YPD+G418. The frequency of BIR was scored as a ratio of G418R ura− colonies to the total number of cells plated (represented by the number of colonies on YPD plates). At least two independently constructed strains for every genotype were used and at least three repeats for each genotype were performed to calculate standard deviations.
Telomeres are the ends of linear eukaryotic chromosomes maintained by an enzyme called telomerase. Non-telomeric DNA ends are often generated as a result of broken replication forks and are usually repaired by break-induced replication (BIR) or homologous recombination to avoid genomic instability. However, telomerase can interfere with the repair by adding a new telomere to a broken DNA end, or the break can be ligated to a telomere, thereby inducing genome re-arrangements that are often found in human genetic disorders and cancer. To understand how cells avoid erroneous repair, we studied cdc9-1 yeast mutant cells that generate broken replication forks with high frequency. We discovered that, in cells with DNA damage, a helicase called Pif1 is phosphorylated and this phosphorylation enables Pif1 not only to inhibit telomerase at broken DNA ends but also stimulate the break repair by BIR, which in turn leads to additional telomere lengthening. Thus, a new regulatory pathway stimulates accurate break repair by BIR and at the same time promotes telomerase activity at telomeres.
biochemistry
telomeres
chromosomes
cell biology
chromosome biology
chromosome structure and function
genetics
biology and life sciences
dna
dna repair
dna recombination
homologous recombination
Break-Induced Replication Requires DNA Damage-Induced Phosphorylation of Pif1 and Leads to Telomere Lengthening