Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres

To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle‐dependent recruitment of telomere‐specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S‐phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase ε (Polε) arrived at telomeres earlier than the lagging strand DNA polymerases α (Polα) and δ (Polδ). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polε, whereas S‐phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polα. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited.     

BLM and SLX4 play opposing roles in recombination‐dependent replication at human telomeres

Alternative lengthening of telomeres (ALT) is a telomere lengthening pathway that predominates in aggressive tumors of mesenchymal origin; however, the underlying mechanism of telomere synthesis is not fully understood. Here, we show that the BLM–TOP3A–RMI (BTR) dissolvase complex is required for ALT‐mediated telomere synthesis. We propose that recombination intermediates formed during strand invasion are processed by the BTR complex, initiating rapid and extensive POLD3‐dependent telomere synthesis followed by dissolution, with no overall exchange of telomeric DNA. This process is counteracted by the SLX4–SLX1–ERCC4 complex, which promotes resolution of the recombination intermediate, resulting in telomere exchange in the absence of telomere extension. Our data are consistent with ALT being a conservative DNA replication process, analogous to break‐induced replication, which is dependent on BTR and counteracted by SLX4 complex‐mediated resolution events.     

Looping‐out mechanism for resolution of replicative stress at telomeres

Repetitive DNA is prone to replication fork stalling, which can lead to genome instability. Here, we find that replication fork stalling at telomeres leads to the formation of t‐circle‐tails, a new extrachromosomal structure that consists of circular telomeric DNA with a single‐stranded tail. Structurally, the t‐circle‐tail resembles cyclized leading or lagging replication intermediates that are excised from the genome by topoisomerase II‐mediated cleavage. We also show that the DNA damage repair machinery NHEJ is required for the formation of t‐circle‐tails and for the resolution of stalled replication forks, suggesting that NHEJ, which is normally constitutively suppressed at telomeres, is activated in the context of replication stress. Inhibition of NHEJ or knockout of DNA‐PKcs impairs telomere replication, leading to multiple‐telomere sites (MTS) and telomere shortening. Collectively, our results support a “looping‐out” mechanism, in which the stalled replication fork is cut out and cyclized to form t‐circle‐tails, and broken DNA is religated. The telomere loss induced by replication stress may serve as a new factor that drives replicative senescence and cell aging.     

Fission yeast Taz1 and RPA are synergistically required to prevent rapid telomere loss

The telomere complex must allow nucleases and helicases to process chromosome ends to make them substrates for telomerase, while preventing these same activities from disrupting chromosome end-protection. Replication protein A (RPA) binds to single-stranded DNA and is required for DNA replication, recombination, repair, and telomere maintenance. In fission yeast, the telomere binding protein Taz1 protects telomeres and negatively regulates telomerase. Here, we show that taz1-d rad11-D223Y double mutants lose their telomeric DNA, indicating that RPA (Rad11) and Taz1 are synergistically required to prevent telomere loss. Telomere loss in the taz1-d rad11-D223Y double mutants was suppressed by additional mutation of the helicase domain in a RecQ helicase (Rqh1), or by overexpression of Pot1, a single-strand telomere binding protein that is essential for protection of chromosome ends. From our results, we propose that in the absence of Taz1 and functional RPA, Pot1 cannot function properly and the helicase activity of Rqh1 promotes telomere loss. Our results suggest that controlling the activity of Rqh1 at telomeres is critical for the prevention of genomic instability.     

Replication Protein A‐1 Has a Preference for the Telomeric G‐rich Sequence in Trypanosoma cruzi

Replication protein A (RPA), the major eukaryotic single‐stranded binding protein, is a heterotrimeric complex formed by RPA‐1, RPA‐2, and RPA‐3. RPA is a fundamental player in replication, repair, recombination, and checkpoint signaling. In addition, increasing evidences have been adding functions to RPA in telomere maintenance, such as interaction with telomerase to facilitate its activity and also involvement in telomere capping in some conditions. Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoa parasite that appears early in the evolution of eukaryotes. Recently, we have showed that T. cruziRPA presents canonical functions being involved with DNA replication and DNA damage response. Here, we found by FISH/IF assays that T. cruziRPA localizes at telomeres even outside replication (S) phase. In vitro analysis showed that one telomeric repeat is sufficient to bind RPA‐1. Telomeric DNA induces different secondary structural modifications on RPA‐1 in comparison with other types of DNA. In addition, RPA‐1 presents a higher affinity for telomeric sequence compared to randomic sequence, suggesting that RPA may play specific roles in T. cruzi telomeric region.     

Mammalian DNA2 helicase/nuclease cleaves G‐quadruplex DNA and is required for telomere integrity

Efficient and faithful replication of telomeric DNA is critical for maintaining genome integrity. The G‐quadruplex (G4) structure arising in the repetitive TTAGGG sequence is thought to stall replication forks, impairing efficient telomere replication and leading to telomere instabilities. However, pathways modulating telomeric G4 are poorly understood, and it is unclear whether defects in these pathways contribute to genome instabilities in vivo. Here, we report that mammalian DNA2 helicase/nuclease recognizes and cleaves telomeric G4 in vitro. Consistent with DNA2's role in removing G4, DNA2 deficiency in mouse cells leads to telomere replication defects, elevating the levels of fragile telomeres (FTs) and sister telomere associations (STAs). Such telomere defects are enhanced by stabilizers of G4. Moreover, DNA2 deficiency induces telomere DNA damage and chromosome segregation errors, resulting in tetraploidy and aneuploidy. Consequently, DNA2‐deficient mice develop aneuploidy‐associated cancers containing dysfunctional telomeres. Collectively, our genetic, cytological, and biochemical results suggest that mammalian DNA2 reduces replication stress at telomeres, thereby preserving genome stability and suppressing cancer development, and that this may involve, at least in part, nucleolytic processing of telomeric G4.     

RPA activates the XPF‐ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks

During replication‐coupled DNA interstrand crosslink (ICL) repair, the XPF‐ERCC1 endonuclease is required for the incisions that release, or “unhook”, ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL. Here, we report that while purified XPF‐ERCC1 incises simple ICL‐containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single‐stranded DNA (ssDNA)‐binding replication protein A (RPA) selectively restores XPF‐ERCC1 endonuclease activity on this structure. The 5′–3′ exonuclease SNM1A can load from the XPF‐ERCC1‐RPA‐induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF‐ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.     

Processing of G4 DNA by Dna2 helicase/nuclease and replication protein A (RPA) provides insights into the mechanism of Dna2/RPA substrate recognition

The polyguanine-rich DNA sequences commonly found at telomeres and in rDNA arrays have been shown to assemble into structures known as G quadruplexes, or G4 DNA, stabilized by base-stacked G quartets, an arrangement of four hydrogen-bonded guanines. G4 DNA structures are resistant to the many helicases and nucleases that process intermediates arising in the course of DNA replication and repair. The lagging strand DNA replication protein, Dna2, has demonstrated a unique localization to telomeres and a role in de novo telomere biogenesis, prompting us to study the activities of Dna2 on G4 DNA-containing substrates. We find that yeast Dna2 binds with 25-fold higher affinity to G4 DNA formed from yeast telomere repeats than to single-stranded DNA of the same sequence. Human Dna2 also binds G4 DNAs. The helicase activities of both yeast and human Dna2 are effective in unwinding G4 DNAs. On the other hand, the nuclease activities of both yeast and human Dna2 are attenuated by the formation of G4 DNA, with the extent of inhibition depending on the topology of the G4 structure. This inhibition can be overcome by replication protein A. Replication protein A is known to stimulate the 5'- to 3'-nuclease activity of Dna2; however, we go on to show that this same protein inhibits the 3'- to 5'-exo/endonuclease activity of Dna2. These observations are discussed in terms of possible roles for Dna2 in resolving G4 secondary structures that arise during Okazaki fragment processing and telomere lengthening.     

Unwinding protein complexes in ALTernative telomere maintenance

Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA‐binding proteins and a number of three dimensional nucleic acid structures including G‐quartets and D‐loops. A number of studies suggest that the BLM and WRN recQ‐like helicases play important roles in recombination‐mediated mechanisms of telomere elongation or A lternative L engthening of T elomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT‐associated nuclear bodies in telomerase‐negative immortalized cell lines and interact with the telomere‐specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double‐strand break repair processes such as non‐homologous end joining, homologous recombination‐mediated repair, resolution of stalled replication forks and synthesis‐dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ‐like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec‐Q‐like helicases and their role in telomere elongation during ALT processes. J. Cell. Biochem. 109: 7–15, 2010. © 2009 Wiley‐Liss, Inc.     

Molecular steps of G‐overhang generation at human telomeres and its function in chromosome end protection

Telomeric G‐overhangs are required for the formation of the protective telomere structure and telomerase action. However, the mechanism controlling G‐overhang generation at human telomeres is poorly understood. Here, we show that G‐overhangs can undergo cell cycle‐regulated changes independent of telomerase activity. G‐overhangs at lagging telomeres are lengthened in S phase and then shortened in late S/G2 because of C‐strand fill‐in, whereas the sizes of G‐overhangs at leading telomeres remain stable throughout S phase and are lengthened in G2/M. The final nucleotides at measurable C‐strands are precisely defined throughout the cell cycle, indicating that C‐strand resection is strictly regulated. We demonstrate that C‐strand fill‐in is mediated by DNA polymerase α (polα) and controlled by cyclin‐dependent kinase 1 (CDK1). Inhibition of CDK1 leads to accumulation of lengthened G‐overhangs and induces telomeric DNA damage response. Furthermore, depletion of hStn1 results in elongation of G‐overhangs and an increase in telomeric DNA damage. Our results suggest that G‐overhang generation at human telomeres is regulated by multiple tightly controlled processes and C‐strand fill‐in is under the control of polα and CDK1.     

FANCJ promotes DNA synthesis through G‐quadruplex structures

Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ^−/− cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes.     

Evolution of CST function in telomere maintenance

Telomeres consist of an elaborate, higher-order DNA architecture, and a suite of proteins that provide protection for the chromosome terminus by blocking inappropriate recombination and nucleolytic attack. In addition, telomeres facilitate telomeric DNA replication by physical interactions with telomerase and the lagging strand replication machinery. The prevailing view has been that two distinct telomere capping complexes evolved, shelterin in vertebrates and a trimeric complex comprised of Cdc13, Stn1 and Ten1 (CST) in yeast. The recent discovery of a CST-like complex in plants and humans raises new questions about the composition of telomeres and their regulatory mechanisms in multicellular eukaryotes. In this review we discuss the evolving functions and interactions of CST components and their contributions to chromosome end protection and DNA replication.Key words: telomere, telomerase, telomere protein, CTC1, STN1, TEN1, OB-fold, arabidopsis, DNA polymerase alpha, RPA     

Two combinatorial patterns of telomere histone marks in plants with canonical and non‐canonical telomere repeats

Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are crucial for the maintenance of genome integrity. In most plants, telomeres consist of conserved tandem repeat units comprising the TTTAGGG motif. Recently, non‐canonical telomeres were described in several plants and plant taxons, including the carnivorous plant Genlisea hispidula (TTCAGG/TTTCAGG), the genus Cestrum (Solanaceae; TTTTTTAGGG), and plants from the Asparagales order with either a vertebrate‐type telomere repeat TTAGGG or Allium genus‐specific CTCGGTTATGGG repeat. We analyzed epigenetic modifications of telomeric histones in plants with canonical and non‐canonical telomeres, and further in telomeric chromatin captured from leaves of Nicotiana benthamiana transiently transformed by telomere CRISPR‐dCas9‐eGFP, and of Arabidopsis thaliana stably transformed with TALE_telo C‐3×GFP. Two combinatorial patterns of telomeric histone modifications were identified: (i) an Arabidopsis‐like pattern (A. thaliana, G. hispidula, Genlisea nigrocaulis, Allium cepa, Narcissus pseudonarcissus, Petunia hybrida, Solanum tuberosum, Solanum lycopersicum) with telomeric histones decorated predominantly by H3K9me2; (ii) a tobacco‐like pattern (Nicotiana tabacum, N. benthamiana, C. elegans) with a strong H3K27me3 signal. Our data suggest that epigenetic modifications of plant telomere‐associated histones are related neither to the sequence of the telomere motif nor to the lengths of the telomeres. Nor the phylogenetic position of the species plays the role; representatives of the Solanaceae family are included in both groups. As both patterns of histone marks are compatible with fully functional telomeres in respective plants, we conclude that the described specific differences in histone marks are not critical for telomere functions.     

FEN1 Ensures Telomere Stability by Facilitating Replication Fork Re-initiation

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.     

Characterization of genetic interactions with RFA1: the role of RPA in DNA replication and telomere maintenance

Replication protein A (RPA) is a heterotrimeric single-stranded DNA binding protein whose role in DNA replication, recombination and repair has been mainly elucidated through in vitro biochemical studies utilizing the mammalian complex. However, the identification of homologs of all three subunits in Saccharomyces cerevisiae offers the opportunity of examining the in vivo role of RPA. In our laboratory, we have previously isolated a missense allele of the RFA1 gene, encoding the p70 subunit of the RPA complex. Strains containing this mutant allele, rfa1-D228Y, display increased levels of direct-repeat recombination, decreased levels of heteroallelic recombination, UV sensitivity and a S-phase delay. In this study, we have characterized further the role of RPA by screening other replication and repair mutants for a synthetic lethal phenotype in combination with the rfa1-D228Y allele. Among the replication mutants examined, only one displayed a synthetic lethal phenotype, pol12-100, a conditional allele of the B subunit of pol alpha-primase. In addition, a delayed senescence phenotype was observed in raf1-D228Y strains containing a null mutation of HDF1, the S. cerevisiae homolog of the 70 kDa subunit of Ku. Interestingly, a synergistic reduction in telomere length observed in the double mutants suggests that the shortening of telomeres may be the cause of the decreased viability in these strains. Furthermore, this result represents the first evidence of a role for RPA in telomere maintenance.     

Multiple genetic pathways regulate replicative senescence in telomerase‐deficient yeast

Most human tissues express low levels of telomerase and undergo telomere shortening and eventual senescence; the resulting limitation on tissue renewal can lead to a wide range of age‐dependent pathophysiologies. Increasing evidence indicates that the decline in cell division capacity in cells that lack telomerase can be influenced by numerous genetic factors. Here, we use telomerase‐defective strains of budding yeast to probe whether replicative senescence can be attenuated or accelerated by defects in factors previously implicated in handling of DNA termini. We show that the MRX (Mre11‐Rad50‐Xrs2) complex, as well as negative (Rif2) and positive (Tel1) regulators of this complex, comprise a single pathway that promotes replicative senescence, in a manner that recapitulates how these proteins modulate resection of DNA ends. In contrast, the Rad51 recombinase, which acts downstream of the MRX complex in double‐strand break (DSB) repair, regulates replicative senescence through a separate pathway operating in opposition to the MRX‐Tel1‐Rif2 pathway. Moreover, defects in several additional proteins implicated in DSB repair (Rif1 and Sae2) confer only transient effects during early or late stages of replicative senescence, respectively, further suggesting that a simple analogy between DSBs and eroding telomeres is incomplete. These results indicate that the replicative capacity of telomerase‐defective yeast is controlled by a network comprised of multiple pathways. It is likely that telomere shortening in telomerase‐depleted human cells is similarly under a complex pattern of genetic control; mechanistic understanding of this process should provide crucial information regarding how human tissues age in response to telomere erosion.     

CTC1‐STN1 coordinates G‐ and C‐strand synthesis to regulate telomere length

Coats plus (CP) is a rare autosomal recessive disorder caused by mutations in CTC1, a component of the CST (CTC1, STN1, and TEN1) complex important for telomere length maintenance. The molecular basis of how CP mutations impact upon telomere length remains unclear. The CP CTC1^L1142H mutation has been previously shown to disrupt telomere maintenance. In this study, we used CRISPR/Cas9 to engineer this mutation into both alleles of HCT116 and RPE cells to demonstrate that CTC1:STN1 interaction is required to repress telomerase activity. CTC1^L1142H interacts poorly with STN1, leading to telomerase‐mediated telomere elongation. Impaired interaction between CTC1^L1142H:STN1 and DNA Pol‐α results in increased telomerase recruitment to telomeres and further telomere elongation, revealing that C:S binding to DNA Pol‐α is required to fully repress telomerase activity. CP CTC1 mutants that fail to interact with DNA Pol‐α resulted in loss of C‐strand maintenance and catastrophic telomere shortening. Our findings place the CST complex as an important regulator of both G‐strand extensions by telomerase and C‐strand synthesis by DNA Pol‐α.     

Budding yeast Rap1, but not telomeric DNA,is inhibitory for multiple stages of DNA replication in vitro

Telomeres are copied and reassembled each cell division cycle through a multistep process called telomere replication. Most telomeric DNA is duplicated semiconservatively during this process, but replication forks frequently pause or stall at telomeres in yeast, mouse and human cells, potentially causing chronic telomere shortening or loss in a single cell cycle. We have investigated the cause of this effect by examining the replication of telomeric templates in vitro. Using a reconstituted assay for eukaryotic DNA replication in which a complete eukaryotic replisome is assembled and activated with purified proteins, we show that budding yeast telomeric DNA is efficiently duplicated in vitro unless the telomere binding protein Rap1 is present. Rap1 acts as a roadblock that prevents replisome progression and leading strand synthesis, but also potently inhibits lagging strand telomere replication behind the fork. Both defects can be mitigated by the Pif1 helicase. Our results suggest that GC-rich sequences do not inhibit DNA replication per se, and that in the absence of accessory factors, telomere binding proteins can inhibit multiple, distinct steps in the replication process.