Asymmetrical end structures of leading and lagging telomeres in Saccharomyces cerevisiae dictate the nature of the end replication problem.

Changes in telomere chromatin have been linked to cellular senescence, but the underlying mechanisms and impact on lifespan are unclear. We found that inactivation of the Sas2 histone acetyltransferase delays senescence in Saccharomyces cerevisiae telomerase (tlc1) mutants through a homologous recombination-dependent mechanism. Sas2 acetylates histone H4 lysine 16 (H4K16), and telomere shortening in tlc1 mutants was accompanied by a selective and Sas2-dependent increase in subtelomeric H4K16 acetylation. Further, mutation of H4 lysine 16 to arginine, which mimics constitutively deacetylated H4K16, delayed senescence and was epistatic to sas2 deletion, indicating that deacetylated H4K16 mediates the delay caused by sas2 deletion. Sas2 normally prevents the Sir2/3/4 heterochromatin complex from leaving the telomere and spreading to internal euchromatic loci. Senescence was delayed by sir3 deletion, but not sir2 deletion, indicating that senescence delay is mediated by release of Sir3 specifically from the telomere repeats. In contrast, sir4 deletion sped senescence and blocked the delay conferred by sas2 or sir3 deletion. We thus show that manipulation of telomere chromatin modulates senescence caused by telomere shortening.

Reverse Transcribing the Code for Chromosome Stability

Telomeres are nucleoprotein structures that protect and maintain the ends of eukaryotic linear chromosomes. Telomeres shorten at each round of DNA replication due to the end replication problem. The enzyme telomerase, by adding telomeric repeats to chromosome ends, can counteract this process. In the absence of telomerase, telomeres progressively shorten until they reach a critical length that activates the DNA damage response, thereby halting the cell cycle in a condition referred to as replicative senescence. Telomeres are transcribed into a long, non-coding RNA dubbed TERRA, which can hybridize with its template strand, thereby forming R-loops at S. cerevisiae and human telomeres. Recent data implicate telomeric R-loops in the promotion of homologous recombination at telomeres, leading to telomere lengthening events which can partially compensate for telomere shortening in the absence of telomerase. Telomeric R-loops are regulated by RNase H1 and H2 enzymes, which can degrade the RNA moiety of RNA-DNA hybrids. While the accumulation of telomeric R-loops in cells lacking both enzymes delays senescence onset by promoting homologous recombination at telomeres, the depletion of telomeric R-loops by overexpressing RNase H1 leads to premature senescence onset. This PhD thesis aims to better understand how telomeric R-loops are regulated especially during replicative senescence in S. cerevisiae. We found that RNase H2 localizes to long telomeres and physically interacts with the telomere-associated protein Rif2, which is required for RNase H2 recruitment to telomeres. Accordingly, in the absence of Rif2 telomeric R-loops accumulate, indicating that Rif2 and RNase H2 play a pivotal role in restricting R-loops at long telomeres. Importantly, the interaction between RNase H2 and Rif2 is strongest in late S phase, which is reflected in the degradation of telomeric R-loops in this time frame. We propose that this cell cycle regulated telomeric R-loop degradation is required to avoid collisions of the replication machinery, which replicates long telomeres in late S phase, with R-loops, an event that could have detrimental effects on telomere stability. It was previously shown that, as telomeres shorten, Rif2 localization to telomeres is diminished. We show that decreased Rif2 association to short telomeres leads to impaired recruitment of RNase H2, which is functionally reflected in the accumulation of R-loops at short telomeres. Moreover, while RNase H1 could not be detected at long telomeres, we observed its localization to short telomeres, thereby indicating a distinct requirement for the RNase H enzymes. By analyzing the effect of single RNase H enzymes deletion on the kinetics of senescence onset in telomerase negative cells, we revealed an opposing effect of the two enzymes, suggesting that, differently from what was proposed, RNase H enzymes do not have redundant functions at telomeres. In conclusion, we propose that, while at long telomeres R-loops are timely regulated by Rif2-RNase H2 to avoid collisions with the replication machinery, at short telomeres R-loops are allowed to accumulate, thereby promoting homologous recombination-mediated telomere extension.

The Rat1p 5′ to 3′ Exonuclease Degrades Telomeric Repeat-Containing RNA and Promotes Telomere Elongation in Saccharomyces cerevisiae

During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared with deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from their active site through a bulky side chain residue that can sterically block the 2'-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase γ discriminates ribonucleotides efficiently but differentially based on the base identity. Whereas UTP is discriminated by 77,000-fold compared with dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme was reduced 3-14-fold, depending on the identity of the incoming nucleotide, when it extended from a primer containing a 3'-terminal ribonucleotide. DNA polymerase γ is also proficient in performing single-nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides, mainly due to its defects in deoxyribonucleoside triphosphate binding, and is also a poor reverse transcriptase. The potential biochemical defects of a patient harboring a disease mutation in the same amino acid (E895G) are discussed.

Human telomeres contain single-stranded 3' G-overhangs that function in telomere end protection and telomerase action. Previously we have demonstrated that multiple steps involving C-strand end resection, telomerase elongation and C-strand fill-in contribute to G-overhang generation in telomerase-positive cancer cells. However, how G-overhangs are generated in telomerase-negative human somatic cells is unknown. Here, we report that C-strand fill-in is present at lagging-strand telomeres in telomerase-negative human cells but not at leading-strand telomeres, suggesting that C-strand fill-in is independent of telomerase extension of G-strand. We further show that while cyclin-dependent kinase 1 (CDK1) positively regulates C-strand fill-in, CDK1 unlikely regulates G-overhang generation at leading-strand telomeres. In addition, DNA polymerase α (Polα) association with telomeres is not altered upon CDK1 inhibition, suggesting that CDK1 does not control the loading of Polα to telomeres during fill-in. In summary, our results reveal that G-overhang generation at leading- and lagging-strand telomeres are regulated by distinct mechanisms in human cells.

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA.DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi-Goutières syndrome. We have determined the crystal structure of the three-protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein-protein interaction motifs that could allow for efficient participation in multiple cellular functions. We have identified four conserved acidic residues in the active site that are necessary for activity and suggest a two-metal ion mechanism of catalysis for RNase H2. An Okazaki fragment has been modeled into the RNase H2 nucleic acid binding site providing insight into the recognition of RNA.DNA junctions by the RNase H2. Further structural and biochemical analyses show that some RNase H2 disease-causing mutations likely result in aberrant protein-protein interactions while the RNase H2A subunit-G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification.

DNA looping is one of the mechanisms involved in telomere maintenance. It probably provides a solution not only to 'the end-replication problem', but also for the protection of chromosomal ends against degradation enzymes and, as typical double-strand breaks, from DNA repair machinery. Telomeric loops (t-loops) formed by an invasion of protruding 3' overhangs into the double-stranded telomeric regions were observed in a variety of organisms ranging from ciliates to mammals. Genetic data indicate that looping also occurs at the telomeres of Saccharomyces cerevisiae, suggesting its importance for telomere function in yeast. However, several observations argue against the presence of 'true' t-loops in the budding yeast telomeres (e.g. the lack of TRF-like protein, heterogeneous telomeric sequences). Instead, telomeres in S. cerevisiae appear to form fold-back structures mediated by protein-protein interactions. To directly visualize the telomeric structure in budding yeast, we developed a system based on a mini-chromosome carrying an array of lac operator sequences allowing its purification by the lac repressor affinity column. In contrast to budding yeast, the fission yeast Schizosaccharomyces pombe contains a homologue of the human telomeric protein TRF2, designated Taz1p. As the TRF2 protein has been implicated in remodelling telomeres into t-loops, the ability of Taz1p to promote t-loop formation is examined by electron microscopy using purified protein and synthetic templates containing a double-stranded fission yeast telomeric tract. Our studies should shed some light not only on telomeric architecture in yeast, but should also be instrumental in deciphering detailed telomeric structure in higher eukaryotes.

Telomeres are terminal repetitive DNA sequences whose stability requires the coordinated actions of telomere-binding proteins and the DNA replication and repair machinery. Recently, we demonstrated that the DNA replication and repair protein Flap endonuclease 1 (FEN1) is required for replication of lagging strand telomeres. Here, we demonstrate for the first time that FEN1 is required for efficient re-initiation of stalled replication forks. At the telomere, we find that FEN1 depletion results in replicative stress as evidenced by fragile telomere expression and sister telomere loss. We show that FEN1 participation in Okazaki fragment processing is not required for efficient telomere replication. Instead we find that FEN1 gap endonuclease activity, which processes DNA structures resembling stalled replication forks, and the FEN1 interaction with the RecQ helicases are vital for telomere stability. Finally, we find that FEN1 depletion neither impacts cell cycle progression nor in vitro DNA replication through non-telomeric sequences. Our finding that FEN1 is required for efficient replication fork re-initiation strongly suggests that the fragile telomere expression and sister telomere losses observed upon FEN1 depletion are the direct result of replication fork collapse. Together, these findings suggest that other nucleases compensate for FEN1 loss throughout the genome during DNA replication but fail to do so at the telomere. We propose that FEN1 maintains stable telomeres by facilitating replication through the G-rich lagging strand telomere, thereby ensuring high fidelity telomere replication.

RNA/DNA hybrids are processed by RNases H1 and H2, while single ribonucleoside-monophosphates (rNMPs) embedded in genomic DNA are removed by the error-free, RNase H2-dependent ribonucleotide excision repair (RER) pathway. In the absence of RER, however, topoisomerase 1 (Top1) can cleave single genomic rNMPs in a mutagenic manner. In RNase H2-deficient mice, the accumulation of genomic rNMPs above a threshold of tolerance leads to catastrophic genomic instability that causes embryonic lethality. In humans, deficiencies in RNase H2 induce the autoimmune disorders Aicardi-Goutières syndrome and systemic lupus erythematosus, and cause skin and intestinal cancers. Recently, we reported that in Saccharomyces cerevisiae, the depletion of Rnr1, the major catalytic subunit of ribonucleotide reductase (RNR), which converts ribonucleotides to deoxyribonucleotides, leads to cell lethality in absence of RNases H1 and H2. We hypothesized that under replicative stress and compromised DNA repair that are elicited by an insufficient supply of deoxyribonucleoside-triphosphates (dNTPs), cells cannot survive the accumulation of persistent RNA/DNA hybrids. Remarkably, we found that cells lacking RNase H2 accumulate ~ 5-fold more genomic rNMPs in absence than in presence of Rnr1. When the load of genomic rNMPs is further increased in the presence of a replicative DNA polymerase variant that over-incorporates rNMPs in leading or lagging strand, cells missing both Rnr1 and RNase H2 suffer from severe growth defects. These are reversed in absence of Top1. Thus, in cells lacking RNase H2 and containing a limiting supply of dNTPs, there is a threshold of tolerance for the accumulation of genomic ribonucleotides that is tightly associated with Top1-mediated DNA damage. In this mini-review, we describe the implications of the loss of RNase H2, or RNases H1 and H2, on the integrity of the nuclear genome and viability of budding yeast cells that are challenged with a critically low supply of dNTPs. We further propose that our findings in budding yeast could pave the way for the study of the potential role of mammalian RNR in RNase H2-related diseases.

Telomeric diseases are a group of rare progeroid genetic syndromes, presenting premature aging phenotypes, characterized for defects on telomere maintenance. In humans, telomeres are heterochromatic structures consisting of long TTAGGG repeats located at the chromosomal ends, which shorten progressively after each DNA replication because of the 'end replication problem'. Critically short telomeres activate a DNA damage response that leads to the arrest of the cell cycle and resulting in cellular senescence or apoptosis. Furthermore, excessively short telomeres are prone to create telomeric fusions, causing genomic instability and malignant transformation. In order to counteract this process, there are two enzymatic complexes, the telomerase complex, with the capacity to elongate telomeres; and the shelterin complex, which protects them from being recognized as DNA breaks. Over the last few decades, several studies have confirmed that critically short telomeres and defects in telomere-associated enzymatic complexes are involved in the development of a group of rare human genetic diseases, with the accumulation of excessive telomere attrition as the underlying cause of these pathologies. Despite the severity of these disorders, there is no curative treatment for any of them. In light of this, this review summarizes the most important defective telomere diseases, their current management, and it presents possible therapeutic strategies based on nanotechnology which may open up new possibilities for their treatment.

During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the alphaH and alphaI helices in the thumb subdomain play in specific RNase H cleavage at the 3'-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3'-end. Further analysis of alphaH mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage.

Eukaryotic genomes are highly complex and divided into linear chromosomes that require end protection from unwarranted fusions, recombination, and degradation in order to maintain genomic stability. This is accomplished through the conserved specialized nucleoprotein structure of telomeres. Due to the repetitive nature of telomeric DNA, and the unusual terminal structure, namely a protruding single stranded 3′ DNA end, completing telomeric DNA replication in a timely and efficient manner is a challenge. For example, the end replication problem causes a progressive shortening of telomeric DNA at each round of DNA replication, thus telomeres eventually lose their protective capacity. This phenomenon is counteracted by the recruitment and the activation at telomeres of the specialized reverse transcriptase telomerase. Despite the importance of telomerase in providing a mechanism for complete replication of telomeric ends, the majority of telomere replication is in fact carried out by the conventional DNA replication machinery. There is significant evidence demonstrating that progression of replication forks is hampered at chromosomal ends due to telomeric sequences prone to form secondary structures, tightly DNA-bound proteins, and the heterochromatic nature of telomeres. The telomeric loop (t-loop) formed by invasion of the 3′-end into telomeric duplex sequences may also impede the passage of replication fork. Replication fork stalling can lead to fork collapse and DNA breaks, a major cause of genomic instability triggered notably by unwanted repair events. Moreover, at chromosomal ends, unreplicated DNA distal to a stalled fork cannot be rescued by a fork coming from the opposite direction. This highlights the importance of the multiple mechanisms involved in overcoming fork progression obstacles at telomeres. Consequently, numerous factors participate in efficient telomeric DNA duplication by preventing replication fork stalling or promoting the restart of a stalled replication fork at telomeres. In this review, we will discuss difficulties associated with the passage of the replication fork through telomeres in both fission and budding yeasts as well as mammals, highlighting conserved mechanisms implicated in maintaining telomere integrity during replication, thus preserving a stable genome.

A BRIEF HISTORY OF TELOMERES AND TELOMERASE Although chromosomes were first observed under the light microscope in the 1880s, it was not until the 1930s that the chromosome end, or telomere, was first appreciated to play a role in the protection from chromosome end-to-end fusions and instability (for review, see Blackburn 1992). In 1978, by chemical sequencing of telomeric DNA from the macronuclei of the ciliate Tetrahymena thermophila , the telomere was found to contain a few dozen repeats of the sequence TTGGGG (Black-burn and Gall 1978); these G-rich telomeric repeats are also conserved in other ciliates (Table 1) (for review, see Henderson 1995). By virtue of the ability of T. thermophila and Oxytricha fallax DNA termini to serve as substrates for “seeding” new telomeres on linear plasmids, telomeres from Saccharomyces cerevisiae were identified and found to comprise a similar G-rich sequence (Szostak and Blackburn 1982; Dani and Zakian 1983; Pluta et al. 1984; Shampay et al. 1984). The characteristic heterogeneous distribution of lengths for a given telomere in T.. thermophila and yeast indicated that telomere lengths vary considerably between chromosome ends within a population (Shampay et al. 1984; Shampay and Blackburn 1988). Despite the limitations imposed by linear templates on DNA replication (termed the end replication problem; see below), telomeres do not shorten in several single-celled organisms upon continuous propagation. This observation, combined with the ability of telomeres to increase in size under certain conditions, led Greider and Blackburn to the eventual discovery of a terminal telomere transferase (telomerase) activity...

Telomerase adds G-rich telomeric repeats to the 3' ends of telomeres1, counteracting telomere shortening caused by loss of telomeric 3' overhangs during leading-strand DNA synthesis ('the end-replication problem'2). Here we report a second end-replication problem that originates from the incomplete duplication of the C-rich telomeric repeat strand (C-strand) by lagging-strand DNA synthesis. This problem is resolved by fill-in synthesis mediated by polymeraseα-primase bound to Ctc1-Stn1-Ten1 (CST-Polα-primase). In vitro, priming for lagging-strand DNA replication does not occur on the 3' overhang and lagging-strand synthesis stops in a zone of approximately 150 nucleotides (nt) more than 26 nt from the end of the template. Consistent with the in vitro data, lagging-end telomeres of cells lacking CST-Polα-primase lost 50-60 nt of telomeric CCCTAA repeats per population doubling. The C-strands of leading-end telomeres shortened by around 100 nt per population doubling, reflecting the generation of 3' overhangs through resection. The measured overall C-strand shortening in the absence of CST-Polα-primase fill-in is consistent with the combined effects of incomplete lagging-strand synthesis and 5' resection at the leading ends. We conclude that canonical DNA replication creates two telomere end-replication problems that require telomerase to maintain the G-rich strand and CST-Polα-primase to maintain the C-strand.