Multiple POT1-TPP1 proteins coat and compact long telomeric single-stranded DNA

Telomeres are nucleoprotein complexes that cap and protect the ends of linear chromosomes. In humans, telomeres end in 50-300 nt of G-rich single-stranded DNA (ssDNA) overhangs. Protection of telomeres 1 (POT1) binds with nanomolar affinity to the ssDNA overhangs and forms a dimer with another telomere-end binding protein called TPP1. Whereas most previous studies utilized telomeric oligonucleotides comprising single POT1-TPP1 binding sites, here, we examined 72- to 144-nt tracts of telomeric DNA containing 6-12 POT1-TPP1 binding sites. Using electrophoretic mobility gel shift assays, size-exclusion chromatography, and electron microscopy, we analyzed telomeric nucleoprotein complexes containing POT1 alone, POT1-TPP1, and a truncated version of POT1 (POT1-N) that maintains its DNA-binding domain. The results revealed that POT1-N and POT1-TPP1 can completely coat long telomeric ssDNA substrates. Furthermore, we show that ssDNA coated with human POT1-TPP1 heterodimers forms compact, potentially ordered structures.     

POT1 proteins in green algae and land plants: DNA-binding properties and evidence of co-evolution with telomeric DNA

Telomeric DNA terminates with a single-stranded 3′ G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1–DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins varied in their minimal DNA-binding sites and nucleotide recognition properties. Green alga POT1 exhibited a strong preference for the canonical plant telomere repeat sequence TTTAGGG with no detectable binding to hexanucleotide telomere repeat TTAGGG found in vertebrates and some plants, including Asparagus. In contrast, POT1 proteins from maize and Asparagus bound TTAGGG repeats with only slightly reduced affinity relative to the TTTAGGG sequence. We conclude that the nucleic acid binding site in plant POT1 proteins is evolving rapidly, and that the recent acquisition of TTAGGG telomere repeats in Asparagus appears to have co-evolved with changes in POT1 DNA sequence recognition.     

Active and Passive Destabilization of G-Quadruplex DNA by the Telomere POT1-TPP1 Complex

Chromosome ends are protected by guanosine-rich telomere DNA that forms stable G-quadruplex (G4) structures. The heterodimeric POT1-TPP1 complex interacts specifically with telomere DNA to shield it from illicit DNA damage repair and to resolve secondary structure that impedes telomere extension. The mechanism by which POT1-TPP1 accomplishes these tasks is poorly understood. Here, we establish the kinetic framework for POT1-TPP1 binding and unfolding of telomere G4 DNA. Our data identify two modes of POT1-TPP1 destabilization of G4 DNA that are governed by protein concentration. At low concentrations, POT1-TPP1 passively captures transiently unfolded G4s. At higher concentrations, POT1-TPP1 proteins bind to G4s to actively destabilize the DNA structures. Cancer-associated POT1-TPP1 mutations impair multiple reaction steps in this process, resulting in less efficient destabilization of G4 structures. The mechanistic insight highlights the importance of cell cycle dependent expression and localization of the POT1-TPP1 complex and distinguishes diverse functions of this complex in telomere maintenance.     

Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding

Human telomeres are composed of duplex TTAGGG repeats and a 3' single-stranded DNA tail. The telomeric DNA is protected and regulated by the shelterin proteins, including the protection of telomeres 1 (POT1) protein that binds telomeric single-stranded DNA. The single-stranded tail can fold into G-quadruplex (G4) DNA. Both POT1 and G4 DNA play important roles in regulating telomere length homeostasis. To date, most studies have focused on individual quadruplexes formed by four TTAGGG repeats. Telomeric tails in human cells have on average six times as many repeats, and no structural studies have examined POT1 binding in competition with G4 DNA folding. Using single molecule atomic force microscopy imaging, we observed that the majority of the telomeric tails of 16 repeats formed two quadruplexes even though four were possible. The result that physiological telomeric tails rarely form the maximum potential number of G4 units provides a structural basis for the coexistence of G4 and POT1 on the same DNA molecule, which is observed directly in the captured atomic force microscopy images. We further observed that POT1 is significantly more effective in disrupting quadruplex DNA on long telomeric tails than an antisense oligonucleotide, indicating a novel POT1 activity beyond simply preventing quadruplex folding.     

DNA-induced dimerization of the single-stranded DNA binding telomeric protein Pot1 from Schizosaccharomyces pombe

Eukaryotic chromosome ends are protected from illicit DNA joining by protein-DNA complexes called telomeres. In most studied organisms, telomeric DNA is composed of multiple short G-rich repeats that end in a single-stranded tail that is protected by the protein POT1. Mammalian POT1 binds two telomeric repeats as a monomer in a sequence-specific manner, and discriminates against RNA of telomeric sequence. While addressing the RNA discrimination properties of SpPot1, the POT1 homolog in Schizosaccharomyces pombe, we found an unanticipated ssDNA-binding mode in which two SpPot1 molecules bind an oligonucleotide containing two telomeric repeats. DNA binding seems to be achieved via binding of the most N-terminal OB domain of each monomer to each telomeric repeat. The SpPot1 dimer may have evolved to accommodate the heterogeneous spacers that occur between S. pombe telomeric repeats, and it also has implications for telomere architecture. We further show that the S. pombe telomeric protein Tpz1, like its mammalian homolog TPP1, increases the affinity of Pot1 for telomeric single-stranded DNA and enhances the discrimination of Pot1 against RNA.     

TPP1 as a versatile player at the ends of chromosomes

Telomeres, the ends of linear eukaryotic chromosomes, are tandem DNA repeats and capped by various telomeric proteins. These nucleoprotein complexes protect telomeres from DNA damage response （DDR）, recombination, and end-to-end fusions, ensuring genome stability. The human telosome/shelterin complex is one of the best-studied telomere-associated protein complexes, made up of six core telomeric proteins TRF1, TRF2, TIN2, RAPI, POT1, and TPPI. TPP1, also known as adrenocortical dysplasia protein homolog （ACD）, is a putative mammalian homolog of TEBP-β and belongs to the oligonucleotide binding （OB）-fold-containing protein family. Three functional domains have been identified within TPP1, the N-terminal OB fold, the POT1 binding recruitment domain （RD）, and the carboxyl-terminal TIN2-interacting domain （TID）. TPP1 can interact with both POT1 and TIN2 to maintain telomere structure, and mediate telomerase recruitment for telomere elongation. These features have indicated TPP1 play an essential role in telomere maintenance. Here, we will review important findings that highlight the functional significance of TPP1, with a focus on its interaction with other telosome components and the telomerase. We will also discuss potential implications in disease therapies.     

Protection of Telomeres 1 Is Required for Telomere Integrity in the Moss Physcomitrella patens

In vertebrates, the single-stranded telomeric DNA binding protein Protection of Telomeres 1 (POT1) shields chromosome ends and prevents them from eliciting a DNA damage response. By contrast, Arabidopsis thaliana encodes two divergent full-length POT1 paralogs that do not exhibit telomeric DNA binding in vitro and have evolved to mediate telomerase regulation instead of chromosome end protection. To further investigate the role of POT1 in plants, we established the moss Physcomitrella patens as a new model for telomere biology and a counterpoint to Arabidopsis. The sequence and architecture of the telomere tract is similar in P. patens and Arabidopsis, but P. patens harbors only a single-copy POT1 gene. Unlike At POT1 proteins, Pp POT1 efficiently bound single-stranded telomeric DNA in vitro. Deletion of the P. patens POT1 gene resulted in the rapid onset of severe developmental defects and sterility. Although telomerase activity levels were unperturbed, telomeres were substantially shortened, harbored extended G-overhangs, and engaged in end-to-end fusions. We conclude that the telomere capping function of POT1 is conserved in early diverging land plants but is subsequently lost in Arabidopsis.     

Multiple hPOT1–TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3′ toward 5′, a feature due to a 3′-end binding preference and to structuring of telomeric DNA

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5′TTAGGG repeats and ends with a 3′ single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1–TPP1 preferentially proceeds from 3′ toward 5′. We explain this directionality in terms of two factors: (i) the preference of hPOT1–TPP1 for the binding site situated at the 3′ end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1–TPP1 in potassium. By comparing binding in K⁺ and in Li⁺, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3′-end and proceeding toward 5′.     

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.     

POT1b protects telomeres from end-to-end chromosomal fusions and aberrant homologous recombination

POT1 (protection of telomere 1) is a highly conserved single-stranded telomeric binding protein that is essential for telomere end protection. Here, we report the cloning and characterization of a second member of the mouse POT family. POT1b binds telomeric DNA via conserved DNA binding oligonucleotide/oligosaccharide (OB) folds. Compared to POT1a, POT1b OB-folds possess less sequence specificity for telomeres. In contrast to POT1a, truncated POT1b possessing only the OB-folds can efficiently localize to telomeres in vivo. Overexpression of a mutant Pot1b allele that cannot bind telomeric DNA initiated a DNA damage response at telomeres that led to p53-dependent senescence. Furthermore, a reduction of the 3' G-rich overhang, increased chromosomal fusions and elevated homologous recombination (HR) were observed at telomeres. shRNA mediated depletion of endogenous Pot1b in Pot1a deficient cells resulted in increased chromosomal aberrations. Our results indicate that POT1b plays important protective functions at telomeres and that proper maintenance of chromosomal stability requires both POT proteins.     

Telomeric D-loops Containing 8-Oxo-2��-deoxyguanosine Are Preferred Substrates for Werner and Bloom Syndrome Helicases and Are Bound by POT1

8-Oxo-2′-deoxyguanosine (8-oxodG) is one of the most important oxidative DNA lesions, and G-rich telomeric DNA is especially susceptible to oxidative DNA damage. RecQ helicases WRN and BLM and telomere-binding protein POT1 are thought to play roles in telomere maintenance. This study examines the ability of WRN, BLM, and RecQ5 to unwind and POT1 to bind telomeric D-loops containing 8-oxodG. The results demonstrate that WRN and BLM preferentially unwind telomeric D-loops containing 8-oxodG and that POT1 binds with higher affinity to telomeric D-loops with 8-oxodG but shows no preference for telomeric single-stranded DNA with 8-oxodG. We speculate that telomeric D-loops with 8-oxodG may have a greater tendency to form G-quadruplex DNA structures than telomeric DNA lacking 8-oxodG.     

Rice proteins that bind single-stranded G-rich telomere DNA

In this work, we have identified and characterized proteins in rice nuclear extracts that specifically bind the single-stranded G-rich telomere sequence. Three types of specific DNA-protein complexes (I, II, and III) were identified by gel retardation assays using synthetic telomere substrates consisting of two or more single-stranded TTTAGGG repeats and rice nuclear extracts. Since each complex has a unique biochemical property and differs in electrophoretic mobility, at least three different proteins interact with the G-rich telomere sequences. These proteins are called rice G-rich telomere binding protein (RGBP) and none of them show binding affinity to double-stranded telomere repeats or single-stranded C-rich sequence. Changing one or two G's to C's in the TTTAGGG repeats abolishes binding activity. RGBPs have a greatly reduced affinity for human and Tetrahymena telomeric sequence and do not efficiently bind the cognate G-rich telomere RNA sequence UUUAGGG. Like other telomere binding proteins, RGBPs are resistant to high salt concentrations. RNase sensitivity of the DNA-protein interactions was tested to investigate whether an RNA component mediates the telomeric DNA-protein interaction. In this assay, we observed a novel complex (complex III) in gel retardation assays which did not alter the mobilities or the band intensities of the two pre-existing complexes (I and II). The complex III, in addition to binding to telomeric sequences, has a binding affinity to rice nuclear RNA, whereas two other complexes have a binding affinity to only single-stranded G-rich telomere DNA. Taken together, these studies suggest that RGBPs are new types of telomere-binding proteins that bind in vitro to single-stranded G-rich telomere DNA in the angiosperms.     

端粒保护蛋白

端粒保护蛋白（pmtection of telomere 1,PoT1）是存在于人和裂殖酵母的端粒相关蛋白,特异性地与端粒单链DNA相结合。人POT1基因位于7号染色体上,由22个外显子组成,其中4个外显子属于跳跃外显子,可形成5个剪接变异体。POT1的功能在于维持端粒的稳定,通过TRF1．TIN2．PIP1-POT通路调节端粒长度。     

POT1 and TRF2 cooperate to maintain telomeric integrity

Mammalian telomeric DNA contains duplex TTAGGG repeats and single-stranded overhangs. POT1 (protection of telomeres 1) is a telomere-specific single-stranded DNA-binding protein, highly conserved in eukaryotes. The biological function of human POT1 is not well understood. In the present study, we demonstrate that POT1 plays a key role in telomeric end protection. The reduction of POT1 by RNA interference led to the loss of telomeric single-stranded overhangs and induced apoptosis, chromosomal instability, and senescence in cells. POT1 and TRF2 interacted with each other to form a complex with telomeric DNA. A dominant negative TRF2, TRF2(DeltaBDeltaM), bound to POT1 and prevented it from binding to telomeres. POT1 overexpression protected against TRF2(DeltaBDeltaM)-induced loss of telomeric single-stranded overhangs, chromosomal instability, and senescence. These results demonstrate that POT1 and TRF2 share in part in the same pathway for telomere capping and suggest that POT1 binds to the telomeric single-stranded DNA in the D-loop and cooperates with TRF2 in t-loop maintenance.     

PTOP interacts with POT1 and regulates its localization to telomeres

Telomere maintenance has been implicated in cancer and ageing, and requires cooperation between a multitude of telomeric factors, including telomerase, TRF1, TRF2, RAP1, TIN2, Tankyrase, PINX1 and POT1 (refs 1-12). POT1 belongs to a family of oligonucleotide-binding (OB)-fold-containing proteins that include Oxytricha nova TEBP, Cdc13, and spPot1, which specifically recognize telomeric single-stranded DNA (ssDNA). In human cells, the loading of POT1 to telomeric ssDNA controls telomerase-mediated telomere elongation. Surprisingly, a human POT1 mutant lacking an OB fold is still recruited to telomeres. However, the exact mechanism by which this recruitment occurs remains unclear. Here we identify a novel telomere protein, PTOP, which interacts with both POT1 and TIN2. PTOP binds to the carboxyl terminus of POT1 and recruits it to telomeres. Inhibition of PTOP by RNA interference (RNAi) or disruption of the PTOP-POT1 interaction hindered the localization of POT1 to telomeres. Furthermore, expression of the respective interaction domains on PTOP and POT1 alone extended telomere length in human cells. Therefore, PTOP heterodimerizes with POT1 and regulates POT1 telomeric recruitment and telomere length.     

POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end

The hallmarks of telomere dysfunction in mammals are reduced telomeric 3' overhangs, telomere fusions, and cell cycle arrest due to a DNA damage response. Here, we report on the phenotypes of RNAi-mediated inhibition of POT1, the single-stranded telomeric DNA-binding protein. A 10-fold reduction in POT1 protein in tumor cells induced neither telomere fusions nor cell cycle arrest. However, the 3' overhang DNA was reduced and all telomeres elicited a transient DNA damage response in G1, indicating that extensive telomere damage can occur without cell cycle arrest or telomere fusions. RNAi to POT1 also revealed its role in generating the correct sequence at chromosome ends. The recessed 5' end of the telomere, which normally ends on the sequence ATC-5', was changed to a random position within the AATCCC repeat. Thus, POT1 determines the structure of the 3' and 5' ends of human chromosomes, and its inhibition generates a novel combination of telomere dysfunction phenotypes in which chromosome ends behave transiently as sites of DNA damage, yet remain protected from nonhomologous end-joining.     

Telomere-end processing: mechanisms and regulation

Telomeres are specialized nucleoprotein complexes that provide protection to the ends of eukaryotic chromosomes. Telomeric DNA consists of tandemly repeated G-rich sequences that terminate with a 3′ single-stranded overhang, which is important for telomere extension by the telomerase enzyme. This structure, as well as most of the proteins that specifically bind double and single-stranded telomeric DNA, are conserved from yeast to humans, suggesting that the mechanisms underlying telomere identity are based on common principles. The telomeric 3′ overhang is generated by different events depending on whether the newly synthesized strand is the product of leading- or lagging-strand synthesis. Here, we review the mechanisms that regulate these processes at Saccharomyces cerevisiae and mammalian telomeres.     

POT1-TPP1 Regulates Telomeric Overhang Structural Dynamics

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Highlights? POT1 binds the telomere overhang sequentially one OB fold at a time ? POT1 binding exhibits 3′ to 5′ directionality ? POT1-TPP1N induces dynamic folding and unfolding of telomeric overhang ? POT1-TPP1N slides back and forth on the telomeric overhang     

Binding of TPP1 Protein to TIN2 Protein Is Required for POT1a,b Protein-mediated Telomere Protection

The single-stranded DNA binding proteins in mouse shelterin, POT1a and POT1b, accumulate at telomeres as heterodimers with TPP1, which binds TIN2 and thus links the TPP1/POT1 dimers with TRF1 and TRF2/Rap1. When TPP1 is tethered to TIN2/TRF1/TRF2, POT1a is thought to block replication protein A binding to the single-stranded telomeric DNA and prevent ataxia telangiectasia and Rad3-related kinase activation. Similarly, TPP1/POT1b tethered to TIN2 can control the formation of the correct single-stranded telomeric overhang. Consistent with this view, the telomeric phenotypes following deletion of POT1a,b or TPP1 are phenocopied in TIN2-deficient cells. However, the loading of TRF1 and TRF2/Rap1 is additionally compromised in TIN2 KO cells, leading to added phenotypes. Therefore, it could not be excluded that, in addition to TIN2, other components of shelterin contribute to the recruitment of TPP1/POT1a,b as suggested by previous reports. To test whether TIN2 is the sole link between TPP1/POT1a,b and telomeres, we defined the TPP1 interaction domain of TIN2 and generated a TIN2 allele that was unable to interact with TPP1 but retained its interaction with TRF1 and TRF2. We demonstrated that cells expressing TIN2ΔTPP1 instead of wild-type TIN2 phenocopy the POT1a,b knockout setting without showing additional phenotypes. Therefore, these results are consistent with TIN2 being the only mechanism by which TPP1/POT1 heterodimers bind to shelterin and function in telomere protection.     

CGGBP1 phosphorylation constitutes a telomere-protection signal

The shelterin proteins are required for telomere integrity. Shelterin dysfunction can lead to initiation of unwarranted DNA damage and repair pathways at chromosomal termini. Interestingly, many shelterin accessory proteins are involved in DNA damage signaling and repair. We demonstrate here that in normal human fibroblasts, telomeric ends are protected by phosphorylation of CGG triplet repeat-binding protein 1 (CGGBP1) at serine 164 (S164). We show that serine 164 is a major phosphorylation site on CGGBP1 with important functions. We provide evidence that one of the kinases that can phosphorylate S164 CGGBP1 is ATR. Overexpression of S164A phospho-deficient CGGBP1 exerted a dominant-negative effect, causing telomeric dysfunction, accelerated telomere shortening, enhanced fusion of telomeres, and crisis. However, overexpression of wild-type or phospho-mimicking S164E CGGBP1 did not cause these effects. This telomere damage was associated with reduced binding of the shelterin protein POT1 to telomeric DNA. Our results suggest that CGGBP1 phosphorylation at S164 is a novel telomere protection signal, which can affect telomere-protective function of the shelterin complex.