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G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding.

ABSTRACT: Human telomeres terminate with a single-stranded 3' G overhang, which can be recognized as a DNA damage site by replication protein A (RPA). The protection of telomeres (POT1)/POT1-interacting protein 1 (TPP1) heterodimer binds specifically to single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded GQ. At 150 mM K(+), POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded. POT1/TPP1 loading blocks RPA's access to both folded and unfolded telomeres by two orders of magnitude. This protection is not observed at 150 mM Na(+), in which ssTEL forms only a less-stable antiparallel GQ. These results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.

SUBMITTER: Ray S

PROVIDER: S-EPMC3939921 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Telomere protection by TPP1/POT1 requires tethering to TIN2.

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POT1-TPP1 regulates telomeric overhang structural dynamics.

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POT1-TPP1 telomere length regulation and disease.

Project description:Telomeres are DNA repeats at the ends of linear chromosomes and are replicated by telomerase, a ribonucleoprotein reverse transcriptase. Telomere length regulation and chromosome end capping are essential for genome stability and are mediated primarily by the shelterin and CST complexes. POT1-TPP1, a subunit of shelterin, binds the telomeric overhang, suppresses ATR-dependent DNA damage response, and recruits telomerase to telomeres for DNA replication. POT1 localization to telomeres and chromosome end protection requires its interaction with TPP1. Therefore, the POT1-TPP1 complex is critical to telomere maintenance and full telomerase processivity. The aim of this mini-review is to summarize recent POT1-TPP1 structural studies and discuss how the complex contributes to telomere length regulation. In addition, we review how disruption of POT1-TPP1 function leads to human disease.

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Structural insights into POT1-TPP1 interaction and POT1 C-terminal mutations in human cancer.

Project description:Mammalian shelterin proteins POT1 and TPP1 form a stable heterodimer that protects chromosome ends and regulates telomerase-mediated telomere extension. However, how POT1 interacts with TPP1 remains unknown. Here we present the crystal structure of the C-terminal portion of human POT1 (POT1C) complexed with the POT1-binding motif of TPP1. The structure shows that POT1C contains two domains, a third OB fold and a Holliday junction resolvase-like domain. Both domains are essential for binding to TPP1. Notably, unlike the heart-shaped structure of ciliated protozoan Oxytricha nova TEBPα-β complex, POT1-TPP1 adopts an elongated V-shaped conformation. In addition, we identify several missense mutations in human cancers that disrupt the POT1C-TPP1 interaction, resulting in POT1 instability. POT1C mutants that bind TPP1 localize to telomeres but fail to repress a DNA damage response and inappropriate repair by A-NHEJ. Our results reveal that POT1 C terminus is essential to prevent initiation of genome instability permissive for tumorigenesis.

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Nonadditivity in the recognition of single-stranded DNA by the schizosaccharomyces pombe protection of telomeres 1 DNA-binding domain, Pot1-DBD.

Project description:The Schizosaccharomyces pombe protection of telomeres 1 (SpPot1) protein recognizes the 3' single-stranded ends of telomeres and provides essential protective and regulatory functions. The ssDNA-binding activity of SpPot1 is conferred by its ssDNA-binding domain, Pot1-DBD (residues 1-389), which can be further separated into two distinct domains, Pot1pN (residues 1-187) and Pot1pC (residues 188-389). Here we show that Pot1pC, like Pot1pN, can function independently of Pot1-DBD and binds specifically to a minimal nonameric oligonucleotide, d(GGTTACGGT), with a K(D) of 400 +/- 70 nM (specifically recognized nucleotides in bold). NMR chemical shift perturbation analysis indicates that the overall structures of the isolated Pot1pN and Pot1pC domains remain intact in Pot1-DBD. Furthermore, alanine scanning reveals modest differences in the ssDNA-binding contacts provided by isolated Pot1pN and within Pot1-DBD. Although the global character of both Pot1pN and Pot1pC is maintained in Pot1-DBD, chemical shift perturbation analysis highlights localized structural differences within the G1/G2 and T3/T4 binding pockets of Pot1pN in Pot1-DBD, which correlate with its distinct ssDNA-binding activity. Furthermore, we find evidence for a putative interdomain interface on Pot1pN that mediates interactions with Pot1pC that ultimately result in the altered ssDNA-binding activity of Pot1-DBD. Together, these data provide insight into the mechanisms underlying the activity and regulation of SpPot1 at the telomere.

| S-EPMC2756145 | biostudies-literature

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