Crystal Structure of the Oxytricha nova Telomere End Binding Protein Complexed with Single Strand DNA

Horvath, Martin P.; Schweiker, Viloya L; Bevilacqua, Joanne M.; Ruggles, James A.; Schultz, Steve C.

doi:10.1016/s0092-8674(00)81720-1

Cited by 252 publications

(293 citation statements)

References 51 publications

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“…Thus, the Cdc13-DBD specificity requirements mirror those observed for the Pot1 proteins, whereby only a subset of the canonical ligand, G 1 X 2 G 3 T 4 , is specifically recognized by Cdc13-DBD. In contrast, TEBPαβ is distinct from the other end-binding proteins in that only two bases in the minimal 12-mer (GGGGTTTTGGGG) produce large affinity changes upon substitution (44,53,54). These affinity changes range from 10 to 80- Structures of the TEBPαβ complex with non-cognate sequences reveal the basis for the observed tolerance for base substitution.…”

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confidence: 98%

Identification of the Determinants for the Specific Recognition of Single-Strand Telomeric DNA by Cdc13

2005

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The single-strand overhang present at all known telomeres plays a critical role in mediating both the capping and telomerase regulation functions of telomeres. The telomere end-binding proteins, Cdc13 in S. cerevisiae, Pot1 in higher eukaryotes, and TEBP in the ciliated protozoa O. nova, exhibit sequence-specific binding to their respective single-strand overhangs. S. cerevisiae telomeres are composed of a heterogeneous mixture of GT-rich telomeric sequence, unlike in higher eukaryotes which have a simple repeat that is maintained with high fidelity. In yeast, the telomeric overhang is recognized by the essential protein Cdc13, which coordinates end-capping and telomerase activities at the telomere. The Cdc13-DNA-binding domain (Cdc13-DBD) binds these telomere sequences with high affinity (3 pM) and sequence specificity. To better understand the basis for this remarkable recognition, we have investigated the binding of the Cdc13-DBD to a series of altered DNA substrates. Although an 11-mer of GT-rich sequence is required for full binding affinity, only 3 of these 11 bases are recognized with high specificity. This specificity differs from that observed in the other known telomere end-binding proteins, but is well suited to the specific role of Cdc13 at yeast telomeres. These studies expand our understanding of telomere recognition by the Cdc13-DBD and of the unique molecular recognition properties of ssDNA binding.Recognition of single-strand DNA (ssDNA) is essential for many fundamental cellular activities, including recombination, repair, replication, transcription, translation, and telomere maintenance. Structural and biochemical studies of the proteins that mediate ssDNA-binding activity have provided key insights into the mechanism of recognition. Generally, small domains containing mixed α/β topologies, such as the OB-fold and KH domain, are used for ssDNA recognition, and binding is achieved through base and backbone interactions that are completely distinct from those used in double-strand DNA recognition (1-5). In many biological contexts DNA needs to be uniformly recognized, thus it is appropriate for this recognition to be non-sequence specific. However, in a few cases, including telomere maintenance, transcription, and viral packaging, it is imperative that recognition be exquisitely sequence specific (3,(6)(7)(8).Telomeres, the DNA, RNA and protein complexes at the ends of chromosomes, play important roles in many essential cellular functions. They regulate the proliferative lifetime of the cell and protect chromosomes from degradation or end-to-end fusion, as well as participate in meiotic segregation and chromatic silencing (reviewed in (9-13)). Telomeres are critical to the maintenance of the genome and normal development. Thus, telomere malfunction can have dramatic adverse impacts on human health, leading to cancer, aging disorders and increased human mortality (14)(15)(16)(17)(18)(19). Therefore, it is essential to understand the mechanisms through which telomeres are properly maintain...

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Identification of the Determinants for the Specific Recognition of Single-Strand Telomeric DNA by Cdc13

2005

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“…Human POT1 (hPOT1) and hPOT1V2 (a splice variant of hPOT1 composed of its DNA-binding domain, used extensively to characterize POT1 structurally and biochemically) bind telomeric DNA with high affinity (K D ∼ 10 nM) and base specificity (10). POT1 is conserved among all mammals and has functional homologs in other species such as Oxytricha nova (12), Tetrahymena thermophila (13), and Schizosaccharomyces pombe (6,14). A sequence-related protein in the plant Arabidopsis thaliana associates with the telomerase ribonucleoprotein rather than the telomeric DNA (15)(16)(17).…”

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confidence: 99%

How telomeric protein POT1 avoids RNA to achieve specificity for single-stranded DNA

Nandakumar

Podell

Cech

2009

Proc. Natl. Acad. Sci. U.S.A.

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The POT1-TPP1 heterodimer, the major telomere-specific singlestranded DNA-binding protein in mammalian cells, protects chromosome ends and contributes to the regulation of telomerase. The recent discovery of telomeric RNA raises the question of how POT1 faithfully binds telomeric ssDNA and avoids illicit RNA binding that could result in its depletion from telomeres. Here we show through binding studies that a single deoxythymidine in a telomeric repeat dictates the DNA versus RNA discrimination by human POT1 and mouse POT1A. We solve the crystal structure of hPOT1 bound to DNA with a ribouridine in lieu of the critical deoxythymidine and show that this substitution results in burying the 2 0 -hydroxyl group in a hydrophobic region (Phe62) of POT1 in addition to eliminating favorable hydrogen-bonding interactions at the POT1-nucleic acid interface. At amino acid 62, Phe discriminates against RNA binding and Tyr allows RNA binding. We further show that TPP1 greatly augments POT1's discrimination against RNA.crystal structure | DNA-protein interaction | POT1-TPP1 | telomere T elomeres are protein-DNA complexes that comprise the termini of linear chromosomes and help maintain the integrity of eukaryotic genomes (1). Telomeric DNA typically consists of a large number of short repeats of dsDNA ending with a singlestranded (ss) G-rich 3 0 overhang (2-4). Specialized telomeric proteins bind to the ds and ss regions of telomeric DNA to prevent inappropriate degradation and fusion events at chromosome ends (5). One such protein, protection of telomeres 1 (POT1), binds specifically to the ss G-rich 3 0 tail of chromosomes (6-11). Human POT1 (hPOT1) and hPOT1V2 (a splice variant of hPOT1 composed of its DNA-binding domain, used extensively to characterize POT1 structurally and biochemically) bind telomeric DNA with high affinity (K D ∼ 10 nM) and base specificity (10). POT1 is conserved among all mammals and has functional homologs in other species such as Oxytricha nova (12), Tetrahymena thermophila (13), and Schizosaccharomyces pombe (6, 14). A sequence-related protein in the plant Arabidopsis thaliana associates with the telomerase ribonucleoprotein rather than the telomeric DNA (15-17).TPP1, another telomeric protein, binds POT1 and is critical for POT1 recruitment to telomeres (18-21). Although human TPP1 (hTPP1) does not bind telomeric DNA directly, it increases the affinity of hPOT1 for telomeric ssDNA (21). Additionally, the hPOT1-hTPP1 complex increases the processivity of telomerase, the unique reverse transcriptase that maintains telomere length by catalyzing the synthesis of telomeric DNA at 3 0 ends of chromosomes (21). hTPP1-N, an N-terminal fragment of hTPP1 that includes an Oligonucleotide/oligosaccharide binding (OB) domain and the POT1-binding domain, fully recapitulates hTPP1's POT1-ssDNA-binding-stimulation and telomerase processivityenhancement functions (21).TERRA is noncoding RNA-containing multiple G-rich telomeric repeats transcribed from chromosome ends (22)(23)(24). TERRA is found in mammals and b...

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“…Chromosomes terminate with a singlestranded overhang of telomeric DNA (29,36). In Oxytricha nova, the telomeric end-binding protein associates with this single-stranded overhang to protect the ultimate chromosome end (18). Recently, POT1 (named for protection of telomeres) was identified as a telomeric end-binding protein homolog in Schizosaccharomyces pombe and in humans (2).…”

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Dynamics of Protein Binding to Telomeres in Living Cells: Implications for Telomere Structure and Function

Mattern¹,

Swiggers²,

Nigg

et al. 2004

Molecular and Cellular Biology

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Telomeric proteins have an essential role in the regulation of the length of the telomeric DNA tract and in protection against end-to-end chromosome fusion. Telomere organization and how individual proteins are involved in different telomere functions in living cells is largely unknown. By using green fluorescent protein tagging and photobleaching, we investigated in vivo interactions of human telomeric DNA-binding proteins with telomeric DNA. Our results show that telomeric proteins interact with telomeres in a complex dynamic fashion: TRF2, which has a dual role in chromosome end protection and telomere length homeostasis, resides at telomeres in two distinct pools. One fraction (ϳ73%) has binding dynamics similar to TRF1 (residence time of ϳ44 s). Interestingly, the other fraction of TRF2 binds with similar dynamics as the putative end-protecting factor hPOT1 (residence time of ϳ11 min). Our data support a dynamic model of telomeres in which chromosome end-protection and telomere length homeostasis are governed by differential binding of telomeric proteins to telomeric DNA.

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Crystal Structure of the Oxytricha nova Telomere End Binding Protein Complexed with Single Strand DNA

Cited by 252 publications

References 51 publications

Identification of the Determinants for the Specific Recognition of Single-Strand Telomeric DNA by Cdc13

Identification of the Determinants for the Specific Recognition of Single-Strand Telomeric DNA by Cdc13

How telomeric protein POT1 avoids RNA to achieve specificity for single-stranded DNA

Dynamics of Protein Binding to Telomeres in Living Cells: Implications for Telomere Structure and Function

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