RPA prevents G‐rich structure formation at lagging‐strand telomeres to allow maintenance of chromosome ends

Replication protein A (RPA) is a highly conserved heterotrimeric single‐stranded DNA‐binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1‐D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging‐strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G‐quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G‐quadruplex. We propose that RPA prevents the formation of G‐quadruplex structures at lagging‐strand telomeres to promote shelterin association and facilitate telomerase action at telomeres.     

Shelterin promotes tethering of late replication origins to telomeres for replication‐timing control

DNA replication initiates at many discrete loci on eukaryotic chromosomes, and individual replication origins are regulated under a spatiotemporal program. However, the underlying mechanisms of this regulation remain largely unknown. In the fission yeast Schizosaccharomyces pombe, the telomere‐binding protein Taz1, ortholog of human TRF1/TRF2, regulates a subset of late replication origins by binding to the telomere‐like sequence near the origins. Here, we showed using a lacO/LacI‐GFP system that Taz1‐dependent late origins were predominantly localized at the nuclear periphery throughout interphase, and were localized adjacent to the telomeres in the G1/S phase. The peripheral localization that depended on the nuclear membrane protein Bqt4 was not necessary for telomeric association and replication‐timing control of the replication origins. Interestingly, the shelterin components Rap1 and Poz1 were required for replication‐timing control and telomeric association of Taz1‐dependent late origins, and this requirement was bypassed by a minishelterin Tpz1‐Taz1 fusion protein. Our results suggest that Taz1 suppresses replication initiation through shelterin‐mediated telomeric association of the origins at the onset of S phase.     

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.     

Sua5p a single‐stranded telomeric DNA‐binding protein facilitates telomere replication

In budding yeast Saccharomyces cerevisiae, telomere length maintenance involves a complicated network as more than 280 telomere maintenance genes have been identified in the nonessential gene deletion mutant set. As a supplement, we identified additional 29 telomere maintenance genes, which were previously taken as essential genes. In this study, we report a novel function of Sua5p in telomere replication. Epistasis analysis and telomere sequencing show that sua5Δ cells display progressively shortened telomeres at early passages, and Sua5 functions downstream telomerase recruitment. Further, biochemical, structural and genetic studies show that Sua5p specifically binds single‐stranded telomeric (ssTG) DNA in vitro through a distinct DNA‐binding region on its surface, and the DNA‐binding ability is essential for its telomere function. Thus, Sua5p represents a novel ssTG DNA‐binding protein and positively regulates the telomere length in vivo.     

Human replication protein A unfolds telomeric G-quadruplexes

G-quadruplex structures inhibit telomerase activity and must be disrupted for telomere elongation during S phase. It has been suggested that the replication protein A (RPA) could unwind and maintain single-stranded DNA in a state amenable to the binding of telomeric components. We show here that under near-physiological in vitro conditions, human RPA is able to bind and unfold G-quadruplex structures formed from a 21mer human telomeric sequence. Analyses by native gel electrophoresis, cross-linking and fluorescence resonance energy transfer indicate the formation of both 1:1 and 2:1 complexes in which G-quadruplexes are unfolded. In addition, quadruplex opening by hRPA is much faster than observed with the complementary DNA, demonstrating that this protein efficiently unfolds G-quartets. A two-step mechanism accounting for the binding of hRPA to G-quadruplexes is proposed. These data point to the involvement of hRPA in regulation of telomere maintenance.     

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.     

Telomeric localization of the Arabidopsis‐type heptamer repeat, (TTTAGGG)n,at the chromosome ends in Saccharina japonica (Phaeophyta)

Telomeres generally consist of short repeats of minisatellite DNA sequences and are useful in chromosome identification and karyotype analysis. To date, telomeres have not been characterized in the economically important brown seaweed Saccharina japonica, thus its full cytogenetic research and genetic breeding potential has not been realized. Herein, the tentative sequence of telomeres in S. japonica was identified by PCR amplification with primers designed based on the Arabidopsis‐type telomere sequence (TTTAGGG)_n, which was chosen out of three possible telomeric repeat DNA sequences typically present in plants and algae. After PCR optimization and cloning, sequence analysis of the amplified products from S. japonica genomic DNA showed that they were composed of repeat units, (TTTAGGG)_n, in which the repeat number ranged from 15 to 63 (n = 46). This type of repeat sequence was verified by a Southern blot assay with the Arabidopsis‐type telomere sequence as a probe. The digestion of S. japonica genomic DNA with the exonuclease Bal31 illustrated that the target sequence corresponding to the Arabidopsis‐type telomere sequence was susceptible to Bal31 digestion, suggesting that the repeat sequence was likely located at the outermost ends of the kelp chromosomes. Fluorescence in situ hybridizations with the aforementioned probe provided the initial cytogenetic evidence that the hybridization signals were principally localized at both ends of S. japonica chromosomes. This study indicates that the telomeric repeat of the kelp chromosomes is (TTTAGGG)_n which differs from the previously reported (TTAGGG)_n sequence in Ectocarpus siliculosus through genome sequencing, thereby suggesting distinct telomeres in brown seaweeds.     

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.     

RPA and POT1

Telomere maintenance in cycling cells relies on both DNA replication and capping by the protein complex shelterin. Two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomere 1 (POT1) play critical roles in DNA replication and telomere capping, respectively. While RPA binds to ssDNA in a non-sequence-specific manner, POT1 specifically recognizes singlestranded TTAGGG telomeric repeats. Loss of POT1 leads to aberrant accumulation of RPA at telomeres and activation of the ataxia telangiectasia and Rad3-related kinase (ATR)-mediated checkpoint response, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. The requirement for both POT1 and RPA in telomere maintenance and the antagonism between the two proteins raises the important question of how they function in concert on telomeric ssDNA. Two interesting models were proposed by recent studies to explain the regulation of POT1 and RPA at telomeres. Here, we discuss how these models help unravel the coordination, and also the antagonism, between POT1 and RPA during the cell cycle.     

RPA and POT1: Friends or foes at telomeres?

Telomere maintenance in cycling cells relies on both DNA replication and capping by the protein complex shelterin. Two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomere 1 (POT1) play critical roles in DNA replication and telomere capping, respectively. While RPA binds to ssDNA in a non-sequence-specific manner, POT1 specifically recognizes singlestranded TTAGGG telomeric repeats. Loss of POT1 leads to aberrant accumulation of RPA at telomeres and activation of the ataxia telangiectasia and Rad3-related kinase (ATR)-mediated checkpoint response, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. The requirement for both POT1 and RPA in telomere maintenance and the antagonism between the two proteins raises the important question of how they function in concert on telomeric ssDNA. Two interesting models were proposed by recent studies to explain the regulation of POT1 and RPA at telomeres. Here, we discuss how these models help unravel the coordination, and also the antagonism, between POT1 and RPA during the cell cycle.Key words: RPA, POT1, telomere, ATR, checkpointTelomeres, the natural ends of chromosomes, are composed of repetitive DNA sequences and “capped” by both specific proteins and non-coding RNAs.^1–3 One of the critical functions of telomeres is to prevent chromosomal ends from recognition by the DNA damage response machinery. Critically short or improperly capped telomeres lead to telomere dysfunction and are a major source of genomic instability.⁴ While telomeres need to be properly capped to remain stable, they also need to be duplicated during each cell division by the DNA replication machinery. The requirement of these two seemingly competing processes for telomere maintenance suggests that the cell must coordinate DNA replication and capping of telomeres to ensure faithful telomere duplication yet avoid an inappropriate DNA damage response.Telomeric DNA is unique in several ways. The bulk of each human telomere is comprised of double-stranded TTA GGG repeats. At the very end of each telomere, a stretch of single-stranded TTAGGG repeats exists as a 3′ overhang. The TTA GGG repeats in the telomeric single-stranded DNA (ssDNA) allow it to loop back and invade telomeric double-stranded DNA (dsDNA), forming a structure called the t-loop.⁵ At the base of the t-loop, the TTAGGG strand of the telomeric dsDNA is displaced by the invading single-stranded 3′ overhang to form a single-stranded D-loop. Thus, the unique DNA sequence and structures of telomeres confer the ability to bind proteins in both sequence- and structure-specific manners, providing the basis for additional regulations.In human cells, telomere capping is orchestrated by the protein complex shelterin, which contains TRF1, TRF2, RAP1, TIN2, TPP1 and POT1.³ Among these shelterin components, TRF1 and TRF2 interact with telomeric dsDNA in a sequence-specific manner, whereas POT1, in a complex with TPP1, binds to telomeric ssDNA in a sequence-specific manner.^6–8 While the human genome contains only one POT1 gene, the mouse genome contains two POT1-related genes, POT1a and POT1b.^9–11 TIN2 functions to stabilize TRF1 and TRF2 DNA binding and also tethers the POT1-TPP1 heterodimer to the rest of the shelterin complex on telomeric dsDNA.^12,13Unlike the properly capped telomeres, double-stranded DNA breaks (DSBs) with ssDNA overhangs are known to activate the ATR checkpoint kinase.^14,15 In a complex with its functional partner ATRIP, ATR is recruited to ssDNA by RPA, a non-sequence-specific ssDNA-binding protein complex.¹⁶ In addition to the ATR-ATRIP kinase complex, several other checkpoint proteins involved in ATR activation are also recruited in the presence of RPA-ssDNA.¹⁵ The structural resemblance between DSBs and telomeres and the presence of ssDNA at telomeres raise the important question as to how ATR activation is repressed at telomeres.     

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.     

Rtt105 functions as a chaperone for replication protein A to preserve genome stability

Generation of single‐stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA‐binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA–ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA–importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA–ssDNA complex. Single‐molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA. These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.     

Replication protein A prevents accumulation of single-stranded telomeric DNA in cells that use alternative lengthening of telomeres

The activation of a telomere maintenance mechanism is required for cancer development in humans. While most tumors achieve this by expressing the enzyme telomerase, a fraction (5–15%) employs a recombination-based mechanism termed alternative lengthening of telomeres (ALT). Here we show that loss of the single-stranded DNA-binding protein replication protein A (RPA) in human ALT cells, but not in telomerase-positive cells, causes increased exposure of single-stranded G-rich telomeric DNA, cell cycle arrest in G2/M phase, accumulation of single-stranded telomeric DNA within ALT-associated PML bodies (APBs), and formation of telomeric aggregates at the ends of metaphase chromosomes. This study demonstrates differences between ALT cells and telomerase-positive cells in the requirement for RPA in telomere processing and implicates the ALT mechanism in tumor cells as a possible therapeutic target.