Joseph Grafton Gall (1928-2024), a founder of modern cell biology, made foundational discoveries on eukaryotic chromosomes and RNA biogenesis. His major contributions include the development of in situ hybridization (later called FISH), demonstration of one DNA double helix/chromosome, isolation of the first eukaryote gene, localization of satellite DNA to centromeric heterochromatin, determination of the first telomeric DNA sequence, and elucidating the structure and functions of Cajal bodies. He was an expert microscopist, a scholar of science history, and an avid naturalist. These attributes, together with his ready embrace of new technologies, contributed to his remarkable success. He was also an early and strong supporter of women in science. His contributions to science and mentoring were recognized by numerous awards including the American Society for Cell Biology's E.B. Wilson Medal, the Society for Developmental Biology's Lifetime Achievement Award, the Albert Lasker Special Achievement Award in Medical Research, and the AAAS Mentor Award for Lifetime Achievement.

RNase P and MRP are highly conserved, multi-protein/RNA complexes with essential roles in processing ribosomal and tRNAs. Three proteins found in both complexes, Pop1, Pop6, and Pop7 are also telomerase-associated. Here, we determine how temperature sensitive POP1 and POP6 alleles affect yeast telomerase. At permissive temperatures, mutant Pop1/6 have little or no effect on cell growth, global protein levels, the abundance of Est1 and Est2 (telomerase proteins), and the processing of TLC1 (telomerase RNA). However, in pop mutants, TLC1 is more abundant, telomeres are short, and TLC1 accumulates in the cytoplasm. Although Est1/2 binding to TLC1 occurs at normal levels, Est1 (and hence Est3) binding is highly unstable. We propose that Pop-mediated stabilization of Est1 binding to TLC1 is a pre-requisite for formation and nuclear localization of the telomerase holoenzyme. Furthermore, Pop proteins affect TLC1 and the RNA subunits of RNase P/MRP in very different ways.

RNase P, an RNA-protein complex, is essential for processing tRNAs. Three of the ten protein subunits of Saccharomyces cerevisiae RNase P (and a related complex, RNase MRP) co-purify with yeast telomerase, another RNA-protein complex. The three telomeraseassociated proteins, Pop1, 6 and 7, bind to TLC1, the RNA subunit of telomerase. In a recent study (Garcia et al. Nat Commun), we used temperature sensitive alleles of the essential POP genes to determine their role in telomerase biogenesis. At permissive temperature, pop mutant cells grow normally, and the abundance of most proteins, including protein subunits of telomerase, is similar to wild type (WT). However, telomeres are short, and the amount of the mature telomerase holoenzyme is low. Unlike the RNA subunit of RNase MRP, TLC1 is more abundant in pop cells and properly folded, except at the Cs2a/TeSS domain where the Pop proteins bind. These defects correlate with defective movement of TLC1 from the cytoplasm, where it associates with telomerase proteins, back to the nucleus where it lengthens telomeres. Thus, Pop proteins are needed for the stable association of telomerase proteins with TLC1, and their reduction sequesters mature telomerase in the cytoplasm, away from its nuclear substrates.

Two common features of centromeres are their transcription into noncoding centromere RNAs (cen-RNAs) and their assembly into nucleosomes that contain a centromere-specific histone H3 (cenH3). Here, we show that Saccharomyces cerevisiae cen-RNA was present in low amounts in wild-type (WT) cells, and that its appearance was tightly cell cycle-regulated, appearing and disappearing in a narrow window in S phase after centromere replication. In cells lacking Cbf1, a centromere-binding protein, cen-RNA was 5-12 times more abundant throughout the cell cycle. In WT cells, cen-RNA appearance occurred at the same time as loss of Cbf1's centromere binding, arguing that the physical presence of Cbf1 inhibits cen-RNA production. Binding of the Pif1 DNA helicase, which happens in mid-late S phase, occurred at about the same time as Cbf1 loss from the centromere, suggesting that Pif1 may facilitate this loss by its known ability to displace proteins from DNA. Cen-RNAs were more abundant in rnh1Δ cells but only in mid-late S phase. However, fork pausing at centromeres was not elevated in rnh1Δ cells but rather was due to centromere-binding proteins, including Cbf1. Strains with increased cen-RNA lost centromere plasmids at elevated rates. In cbf1Δ cells, where both the levels and the cell cycle-regulated appearance of cen-RNA were disrupted, the timing and levels of cenH3 centromere binding were perturbed. Thus, cen-RNAs are highly regulated, and disruption of this regulation correlates with changes in centromere structure and function.

An R-loop is a structure that forms when an RNA transcript stays bound to the DNA strand that encodes it and leaves the complementary strand exposed as a loop of single stranded DNA. R-loops accumulate when the processing of RNA transcripts is impaired. The failure to remove these RNA-DNA hybrids can lead to replication fork stalling and genome instability. Resolution of R-loops is thought to be mediated mainly by RNase H enzymes through the removal and degradation of the RNA in the hybrid. However, DNA helicases can also dismantle R-loops by displacing the bound RNA. In particular, the Pif1 family DNA helicases have been shown to regulate R-loop formation at specific genomic loci, such as tRNA genes and centromeres. Here we review the roles of Pif1 family helicases in vivo and in vitro and discuss evidence that Pif1 family helicases act on RNA-DNA hybrids and highlight their potential roles in complementing RNase H for R-loop resolution.

Pif1 family helicases are found in virtually all eukaryotes. Saccharomyces cerevisiae (Sc) encodes two Pif1 family helicases, ScPif1 and Rrm3. ScPif1 is multifunctional, required not only for maintenance of mitochondrial DNA but also for multiple distinct nuclear functions. Rrm3 moves with the replication fork and promotes movement of the fork through ∼1400 hard-to-replicate sites, including centromeres. Here we show that ScPif1, like Rrm3, bound robustly to yeast centromeres but only if the centromere was active. While Rrm3 binding to centromeres occurred in early to mid S phase, about the same time as centromere replication, ScPif1 binding occurred later in the cell cycle when replication of most centromeres is complete. However, the timing of Rrm3 and ScPif1 centromere binding was altered by the absence of the other helicase, such that Rrm3 centromere binding occurred later in pif1-m2 cells and ScPif1 centromere binding occurred earlier in rrm3Δ cells. As shown previously, the modest pausing of replication forks at centromeres seen in wild-type cells was increased in the absence of Rrm3. While a lack of ScPif1 did not result in increased fork pausing at centromeres, pausing was even higher in rrm3Δ pif1Δ cells than in rrm3Δ cells. Likewise, centromere function as monitored by the loss rate of a centromere plasmid was increased in rrm3Δ but not pif1Δ cells, and was even higher in rrm3Δ pif1Δ cells than in rrm3Δ cells. Thus, ScPif1 promotes centromere replication and segregation, but only in the absence of Rrm3. These data also hint at a potential post-S phase function for ScPif1 at centromeres. These studies add to the growing list of ScPif1 functions that promote chromosome stability.

Pif1 family DNA helicases are conserved from bacteria to humans and have critical and diverse functions in vivo that promote genome integrity. Pif1 family helicases share a 23 amino acid region, called the Pif1 signature motif (SM) that is unique to this family. To determine the importance of the SM, we did mutational and functional analysis of the SM from the Saccharomyces cerevisiae Pif1 (ScPif1). The mutations deleted portions of the SM, made one or multiple single amino acid changes in the SM, replaced the SM with its counterpart from a bacterial Pif1 family helicase and substituted an α-helical domain from another helicase for the part of the SM that forms an α helix. Mutants were tested for maintenance of mitochondrial DNA, inhibition of telomerase at telomeres and double strand breaks, and promotion of Okazaki fragment maturation. Although certain single amino acid changes in the SM can be tolerated, the presence and sequence of the ScPif1 SM were essential for all tested in vivo functions. Consistent with the in vivo analyses, in vitro studies showed that the presence and sequence of the ScPif1 SM were critical for ATPase activity but not substrate binding.

About 25% of global greenhouse gas emissions come from sources in China, more than the United States (14%) and the European Union (10%) combined ([ 1 ][1]). Given China's impact on the climate and the Trump Administration's retreat on international action, we should not only welcome positive signs of China's leadership on climate (“China can lead on climate change,” C. Wang and F. Wang, Letters, 25 August, p. [764][2]) but acknowledge that the world is depending it. We must also demand that China's climate actions be sufficiently quick, genuinely effective, open to scrutiny, and free from diplomatic brinkmanship. Recent declines in energy-related carbon emissions and coal consumption, as well as leadership in manufacturing and deployment of renewable energy technologies, are positive signs, but China's path forward is largely dependent on the effectiveness of its proposed national emissions trading scheme. If effective, this scheme should systematically facilitate China's transition to a low-carbon economy and go a long way toward limiting global climate change. Lessons from other countries, such as those involved in the European Union Emissions Trading Scheme, have shown that emissions trading schemes only work if carbon accounts are accurate, emissions reduction targets are sufficiently ambitious, and the number of free permits allowed is minimized. Furthermore, the supply of offsets cannot distort emissions reduction objectives, and liable entities must be aware of and able to implement their abatement options ([ 2 ][3]–[ 4 ][4]). A trading scheme's effectiveness is ultimately determined by its design architecture and transition trajectory (i.e., the pathway and rate by which an economy transitions from carbon-intensive to low carbon). The seven pilot trading schemes in China over the past few years gave away too many free permits to be truly effective in the short term ([ 5 ][5]). China's national emission trading scheme must strike the elusive balance between a prudently-timed implementation and the need to hastily achieve real emissions reductions. If this can be done, then China most certainly deserves recognition as the world's leading nation on climate action. 1. [↵][6]1. T. Boden, 2. G. Marland, 3. R. J. Andres , “National CO2 emissions from fossil-fuel burning, cement manufacture, and gas flaring: 1751–2014” (Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, U.S. Department of Energy, 2017). 2. [↵][7]1. C. De Perthius, 2. R. Trotignon , Energy Pol. 75, 100 (2014). [OpenUrl][8] 3. 1. R. G. et al ., Science 343, 1316 (2014). [OpenUrl][9][Abstract/FREE Full Text][10] 4. [↵][11]1. R. Schmalensee, 2. R. Stavins , Rev. Environ. Econ. Pol. 11, 59 (2017). [OpenUrl][12] 5. [↵][13]1. L. Xiong et al ., Appl. Energy 185, 1849 (2017). [OpenUrl][14] [1]: #ref-1 [2]: pending:yes [3]: #ref-2 [4]: #ref-4 [5]: #ref-5 [6]: #xref-ref-1-1 "View reference 1 in text" [7]: #xref-ref-2-1 "View reference 2 in text" [8]: {openurl}?query=rft.jtitle%253DEnergy%2BPol.%26rft.volume%253D75%26rft.spage%253D100%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [9]: {openurl}?query=rft.jtitle%253DScience%26rft.stitle%253DScience%26rft.aulast%253DNewell%26rft.auinit1%253DR.%2BG.%26rft.volume%253D343%26rft.issue%253D6177%26rft.spage%253D1316%26rft.epage%253D1317%26rft.atitle%253DCarbon%2BMarket%2BLessons%2Band%2BGlobal%2BPolicy%2BOutlook%26rft_id%253Dinfo%253Adoi%252F10.1126%252Fscience.1246907%26rft_id%253Dinfo%253Apmid%252F24653022%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [10]: /lookup/ijlink/YTozOntzOjQ6InBhdGgiO3M6MTQ6Ii9sb29rdXAvaWpsaW5rIjtzOjU6InF1ZXJ5IjthOjQ6e3M6ODoibGlua1R5cGUiO3M6NDoiQUJTVCI7czoxMToiam91cm5hbENvZGUiO3M6Mzoic2NpIjtzOjU6InJlc2lkIjtzOjEzOiIzNDMvNjE3Ny8xMzE2IjtzOjQ6ImF0b20iO3M6MjM6Ii9zY2kvMzU3LzYzNTYvMTEwNi5hdG9tIjt9czo4OiJmcmFnbWVudCI7czowOiIiO30= [11]: #xref-ref-4-1 "View reference 4 in text" [12]: {openurl}?query=rft.jtitle%253DRev.%2BEnviron.%2BEcon.%2BPol.%26rft.volume%253D11%26rft.spage%253D59%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [13]: #xref-ref-5-1 "View reference 5 in text" [14]: {openurl}?query=rft.jtitle%253DAppl.%2BEnergy%26rft.volume%253D185%26rft.spage%253D1849%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx

Saccharomyces cerevisiae encodes two Pif1 family DNA helicases, Pif1 and Rrm3. Rrm3 promotes DNA replication past stable protein complexes at tRNA genes (tDNAs). We identify a new role for the Pif1 helicase: promotion of replication and suppression of DNA damage at tDNAs. Pif1 binds multiple tDNAs, and this binding is higher in rrm3Δ cells. Accumulation of replication intermediates and DNA damage at tDNAs is higher in pif1Δ rrm3Δ than in rrm3Δ cells. DNA damage at tDNAs in the absence of these helicases is suppressed by destabilizing R-loops while Pif1 and Rrm3 binding to tDNAs is increased upon R-loop stabilization. We propose that Rrm3 and Pif1 promote genome stability at tDNAs by displacing the stable multi-protein transcription complex and by removing R-loops. Thus, we identify tDNAs as a new source of R-loop-mediated DNA damage. Given their large number and high transcription rate, tDNAs may be a potent source of genome instability.

The addition of telomeric DNA to chromosome ends is an essential cellular activity that compensates for the loss of genomic DNA that is due to the inability of the conventional DNA replication apparatus to duplicate the entire chromosome. The telomerase reverse transcriptase and its associated RNA bind to the very end of the telomere via a sequence in the RNA and specific protein-protein interactions. Telomerase RNA also provides the template for addition of new telomeric repeats by the reverse-transcriptase protein subunit. In addition to the template, there are 3 other conserved regions in telomerase RNA that are essential for normal telomerase activity. Here we briefly review the conserved core regions of telomerase RNA and then focus on a recent study in fission yeast that determined the function of another conserved region in telomerase RNA called the Stem Terminus Element (STE).(1) The STE is distant from the templating core of telomerase in both the linear and RNA secondary structure, but, nonetheless, affects the fidelity of telomere sequence addition and, in turn, the ability of telomere binding proteins to bind and protect chromosome ends. We will discuss possible mechanisms of STE action and the suitability of the STE as an anti-cancer target.

Replicative DNA helicases expose the two strands of the double helix to the replication apparatus, but accessory helicases are often needed to help forks move past naturally occurring hard-to-replicate sites, such as tightly bound proteins, RNA/DNA hybrids, and DNA secondary structures. Although the Schizosaccharomyces pombe 5'-to-3' DNA helicase Pfh1 is known to promote fork progression, its genomic targets, dynamics, and mechanisms of action are largely unknown. Here we address these questions by integrating genome-wide identification of Pfh1 binding sites, comprehensive analysis of the effects of Pfh1 depletion on replication and DNA damage, and proteomic analysis of Pfh1 interaction partners by immunoaffinity purification mass spectrometry. Of the 621 high confidence Pfh1-binding sites in wild type cells, about 40% were sites of fork slowing (as marked by high DNA polymerase occupancy) and/or DNA damage (as marked by high levels of phosphorylated H2A). The replication and integrity of tRNA and 5S rRNA genes, highly transcribed RNA polymerase II genes, and nucleosome depleted regions were particularly Pfh1-dependent. The association of Pfh1 with genomic integrity at highly transcribed genes was S phase dependent, and thus unlikely to be an artifact of high transcription rates. Although Pfh1 affected replication and suppressed DNA damage at discrete sites throughout the genome, Pfh1 and the replicative DNA polymerase bound to similar extents to both Pfh1-dependent and independent sites, suggesting that Pfh1 is proximal to the replication machinery during S phase. Consistent with this interpretation, Pfh1 co-purified with many key replisome components, including the hexameric MCM helicase, replicative DNA polymerases, RPA, and the processivity clamp PCNA in an S phase dependent manner. Thus, we conclude that Pfh1 is an accessory DNA helicase that interacts with the replisome and promotes replication and suppresses DNA damage at hard-to-replicate sites. These data provide insight into mechanisms by which this evolutionarily conserved helicase helps preserve genome integrity.

It is widely appreciated that the ends of linear DNA molecules cannot be fully replicated by the conventional replication apparatus. Less well known is that semi-conservative replication of telomeric DNA also presents problems for DNA replication. These problems likely arise from the atypical chromatin structure of telomeres, the GC-richness of telomeric DNA that makes it prone to forming DNA secondary structures, and from RNA-DNA hybrids, formed by transcripts of one or both DNA strands. Given the different aspects of telomeres that complicate their replication, it is not surprising that multiple DNA helicases promote replication of telomeric DNA. This review focuses on one such class of DNA helicases, the Pif1 family of 5′–3′ DNA helicases. In budding and fission yeasts, Pif1 family helicases impact both telomerase-mediated and semi-conservative replication of telomeric DNA as well as recombination-mediated telomere lengthening.

Helicases are nucleic acid-dependent ATPases which can bind and remodel nucleic acids, protein-nucleic acid complexes, or both. They are involved in almost every step in cells related to nucleic acid metabolisms, including DNA replication and repair, transcription, RNA maturation and splicing, and nuclear export processes. Using single-molecule fluorescence-force spectroscopy, we have previously directly observed helicase translocation on long single-stranded DNA and revealed that two monomers of UvrD helicase are required for the initiation of unwinding function. Here, we present the details of fluorescence-force spectroscopy instrumentation, calibration, and activity assays in detail for observing the biochemical activities of helicases in real time and revealing how mechanical forces are involved in protein-nucleic acid interaction. These single-molecule approaches are generally applicable to many other protein-nucleic acid systems.

Telomerase is a specialized reverse transcriptase that maintains the ends of chromosomes in almost all eukaryotes. The core of telomerase consists of telomerase RNA and the reverse transcriptase that uses a short segment without the RNA to template the addition of telomeric repeats. In addition, one or more accessory proteins are required for telomerase action in vivo. The best-studied accessory protein is Est1, which is conserved from yeasts to humans. In budding yeast, Est1 has two critical in vivo functions: By interaction with Cdc13, a telomere-binding protein, it recruits telomerase to telomeres, and it also increases telomerase activity. Although budding yeast telomerase is highly regulated by the cell cycle, Est1 is the only telomerase subunit whose abundance is cell cycle-regulated. Close to 400 yeast genes are reported to affect telomere length, although the specific function of most of them is unknown. With the goal of identifying novel telomerase regulators by mass spectrometry, we developed methods for purifying yeast telomerase and its associated proteins. We summarize the methods we used and describe the experiments that show that four telomerase-associated proteins identified by mass spectrometry, none of which had been linked previously to telomeres, affect telomere length and cell cycle regulation of telomerase by controlling Est1 abundance.

Telomerase, the enzyme that maintains telomeres, preferentially lengthens short telomeres. The S. cerevisiae Pif1 DNA helicase inhibits both telomerase-mediated telomere lengthening and de novo telomere addition at double strand breaks (DSB). Here, we report that the association of the telomerase subunits Est2 and Est1 at a DSB was increased in the absence of Pif1, as it is at telomeres, suggesting that Pif1 suppresses de novo telomere addition by removing telomerase from the break. To determine how the absence of Pif1 results in telomere lengthening, we used the single telomere extension assay (STEX), which monitors lengthening of individual telomeres in a single cell cycle. In the absence of Pif1, telomerase added significantly more telomeric DNA, an average of 72 nucleotides per telomere compared to the 45 nucleotides in wild type cells, and the fraction of telomeres lengthened increased almost four-fold. Using an inducible short telomere assay, Est2 and Est1 no longer bound preferentially to a short telomere in pif1 mutant cells while binding of Yku80, a telomere structural protein, was unaffected by the status of the PIF1 locus. Two experiments demonstrate that Pif1 binding is affected by telomere length: Pif1 (but not Yku80) -associated telomeres were 70 bps longer than bulk telomeres, and in the inducible short telomere assay, Pif1 bound better to wild type length telomeres than to short telomeres. Thus, preferential lengthening of short yeast telomeres is achieved in part by targeting the negative regulator Pif1 to long telomeres.

The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex.

The “end replication problem,” the inability of conventional DNA polymerases to duplicate the very ends of linear DNA molecules, is usually solved by telomerase, a specialized reverse transcriptase. The telomerase holoenzyme consists of the catalytic subunit (TERT), a telomerase RNA (TER) and species-specific accessory factors. Although telomerase RNAs vary enormously in size and nucleotide sequence, 4 structures are highly conserved: (1) a telomere repeat templating sequence, which is single-stranded in the folded RNA; (2) a template boundary element (TBE) helix that is immediately adjacent to the template and prevents synthesis past the template; (3) a pseudoknot that affects template usage, and (4) the stem terminus element (STE), which was discovered in mammals in 2000 where it is essential for telomerase activity in vitro and in vivo yet its mode of action was previously unknown. Except for the STE, the 3 other elements are in the catalytic core region of the RNA. This review summarizes a recent paper that takes advantage of the conservation of STE sequence and structure in the fission yeast Schizosaccharomyces pombe (Fig. 1) to reveal the function of the STE. To understand how the STE works, we generated a panel of fission yeast STE mutants and determined their effects on telomere length. This characterization showed that, as in mammals, the fission yeast STE is essential for telomerase activity and telomere maintenance and identified a partial-loss-of-function allele, ter1-STEloop. In this mutant, the 4 tandem uridines in the STE tetraloop are changed to adenines (Fig. 1). Because telomeres are short but stable in ter1-STEloop cells, the mutant was ideal for analyzing STE function in vivo. Although non-canonical telomere repeats are seen in most organisms, fission yeast telomeric DNA is particularly heterogeneous, as reflected in its consensus sequence, 50-(G)0–6GGTTACAC-30 (the rare cytosine, discussed below, is underlined). Our ability to isolate the ter1-STEloop allele was probably facilitated by this heterogeneity, which arises from the inefficiency of the fission yeast TBE, which is less capable than TBEs in other organisms at preventing run-on reverse transcription. In wild type fission yeast, occasional TBE failure results in reverse transcription into the TBE, which introduces a rare cytosine at the end of the core telomere sequence in about 12% of repeats. The variable number of guanine residues is generated by “stuttering” that arises from the absence of base-pairing between the RNA template and the telomeric DNA end. Because incorporation of the rare cytosine changes the template RNA-telomeric DNA register, its addition results in reduced stuttering and hence fewer guanine-tracts (Reviewed in). Although we considered that the STE promotes holoenzyme formation, TERT association with TER1 was unaffected in ter1-STEloop cells. Likewise, the physical interaction between telomerase and the telomere was normal. However, as in humans, telomerase catalysis is compromised as extracts from ter1-STEloop cells had very low activity. We isolated and cloned telomeric DNA from mutant and wild type cells and determined their sequence. For cloning, we developed a novel method that exploited the rapid but reversible telomere shortening that occurs by growing fission yeast cells at higher temperatures. This approach ensured that we analyzed only the sequence templated by TER1-STEloop. Surprisingly, ter1-STEloop telomeric DNA does not have wild type sequence. Rather, the mutant telomeres have a much higher frequency of rare cytosines and reduced frequency of guanine tracts, 2 phenotypes first seen in cells with mutant TBEs (Fig. 1). The unusual sequence of ter1-STEloop telomeric DNA was recombination independent but telomerase dependent. Thus, the STE affects how the telomerase catalytic core templates nucleotide addition. Because the ter1-STEloop mutation affects the sequence of telomeric DNA in the same way as mutations that disrupt TBE function, we conclude that the STE enforces TBE activity. The TER1-STEloop templated change in telomeric sequence also affected telomeric chromatin. In fission yeast and mammals, telomeres are bound by the multi-subunit shelterin complex, which together with the telomeric DNA, protects telomeres and mediates all telomere functions. Using chromatin immunoprecipitaton, we analyzed telomere binding of

The multifunctional Saccharomyces cerevisiae Pif1 DNA helicase affects the maintenance of telomeric, ribosomal, and mitochondrial DNAs, suppresses DNA damage at G-quadruplex motifs, influences the processing of Okazaki fragments, and promotes breakage induced replication. All of these functions require the ATPase/helicase activity of the protein. Owing to Pif1's critical role in the maintenance of mitochondrial DNA, pif1Δ strains quickly generate respiratory deficient cells and hence grow very slowly. This slow growth makes it difficult to carry out genome-wide synthetic genetic analysis in this background. Here, we used a partial loss of function allele of PIF1, pif1-m2, which is mitochondrial proficient but has reduced abundance of nuclear Pif1. Although pif1-m2 is not a null allele, pif1-m2 cells exhibit defects in telomere maintenance, reduced suppression of damage at G-quadruplex motifs and defects in breakage induced replication. We performed a synthetic screen to identify nonessential genes with a synthetic sick or lethal relationship in cells with low abundance of nuclear Pif1. This study identified eleven genes that were synthetic lethal (APM1, ARG80, CDH1, GCR1, GTO3, PRK1, RAD10, SKT5, SOP4, UMP1, and YCK1) and three genes that were synthetic sick (DEF1, YIP4, and HOM3) with pif1-m2.

Almost 400 genes affect yeast telomere length, including Est1 , which is critical for recruitment and activation of telomerase. Here we use mass spectrometry to identify novel telomerase regulators by their co-purification with the telomerase holoenzyme. In addition to all known subunits, over 100 proteins are telomerase associated, including all three subunits of the essential Cdc48-Npl4-Ufd1 complex as well as three E3 ubiquitin ligases. The Cdc48 complex is evolutionarily conserved and targets ubiquitinated proteins for degradation. Est1 levels are ∼40-fold higher in cells with reduced Cdc48, yet, paradoxically, telomeres are shorter. Furthermore, Est1 is ubiquitinated and its cell cycle-regulated abundance is lost in Cdc48-deficient cells. Deletion of the telomerase-associated E3 ligase, Ufd4, in cdc48-3 cells further increases Est1 abundance but suppresses the telomere length phenotype of the single mutant. These data argue that, in concert with Ufd4, the Cdc48 complex regulates telomerase by controlling the level and activity of Est1.