POT1-TPP1 Binding and Unfolding of Telomere DNA Discriminates against Structural Polymorphism.

Telomeres are nucleoprotein complexes that reside at the ends of linear chromosomes and help maintain genomic integrity. Protection of telomeres 1 (POT1) and TPP1 are telomere-specific proteins that bind as a heterodimer to single-stranded telomere DNA to prevent illicit DNA damage responses and to enhance telomerase-mediated telomere extension. Telomere DNA is guanosine rich and, as such, can form highly stable secondary structures including G-quadruplexes. G-quadruplex DNA folds into different topologies that are determined by several factors including monovalent ion composition and the precise sequence and length of the DNA. Here, we explore the influence of DNA secondary structure on POT1-TPP1 binding. Equilibrium binding assays reveal that the POT1-TPP1 complex binds G-quadruplex structures formed in buffers containing Na(+) with an affinity that is fivefold higher than for G-quadruplex structures formed in the presence of K(+). However, the binding of the second heterodimer is insensitive to DNA secondary structure, presumably due to unfolding resulting from binding of the first POT1-TPP1. We further show that the rate constant for POT1-TPP1-induced unfolding of DNA secondary structure is substantially faster for G-quadruplex topologies formed in the presence of Na(+) ions. When bound to DNA, POT1-TPP1 forms complexes with similar CD spectra and enhances telomerase activity for all DNA substrates tested, regardless of the substrate secondary structure or solution monovalent ion composition. Together, these data indicate that binding of POT1-TPP1 unfolds telomere secondary structure to assist loading of additional heterodimers and to ensure efficient promotion of telomerase-mediated extension.

Multiple facets of TPP1 in telomere maintenance.

Telomeres are nucleoprotein complexes that cap the ends of all linear chromosomes and function to prevent aberrant repair and end-to-end chromosome fusions. In somatic cells, telomere shortening is a natural part of the aging process as it occurs with each round of cell division. In germ and stem cells, however, the enzyme telomerase synthesizes telomere DNA to counter-balance telomere shortening and help maintain cellular proliferation. Of the primary telomere end-binding proteins, TPP1 has recently emerged as a primary contributor in protecting telomere DNA and in recruiting telomerase to the telomere ends. In this review, we summarize the current knowledge regarding the role of TPP1 in telomere maintenance.

Coordinated interactions of multiple POT1-TPP1 proteins with telomere DNA.

Telomeres are macromolecular nucleoprotein complexes that protect the ends of eukaryotic chromosomes from degradation, end-to-end fusion events, and from engaging the DNA damage response. However, the assembly of this essential DNA-protein complex is poorly understood. Telomere DNA consists of the repeated double-stranded sequence 5'-TTAGGG-3' in vertebrates, followed by a single-stranded DNA overhang with the same sequence. Both double- and single-stranded regions are coated with high specificity by telomere end-binding proteins, including POT1 and TPP1, that bind as a heterodimer to single-stranded telomeric DNA. Multiple POT1-TPP1 proteins must fully coat the single-stranded telomere DNA to form a functional telomere. To better understand the mechanism of multiple binding, we mutated or deleted the two guanosine nucleotides residing between adjacent POT1-TPP1 recognition sites in single-stranded telomere DNA that are not required for multiple POT1-TPP1 binding events. Circular dichroism demonstrated that spectra from the native telomere sequence are characteristic of a G-quadruplex secondary structure, whereas the altered telomere sequences were devoid of these signatures. The altered telomere strands, however, facilitated more cooperative loading of multiple POT1-TPP1 proteins compared with the wild-type telomere sequence. Finally, we show that a 48-nucleotide DNA with a telomere sequence is more susceptible to nuclease digestion when coated with POT1-TPP1 proteins than when it is left uncoated. Together, these data suggest that POT1-TPP1 binds telomeric DNA in a coordinated manner to facilitate assembly of the nucleoprotein complexes into a state that is more accessible to enzymatic activity.

Characterization of hibernating ribosomes in mammalian cells.

Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.

Recruitment of Uev1B to Hrs-containing endosomes and its effect on endosomal trafficking.

Endocytosis of signaling receptors, such as epidermal growth factor receptor (EGFR), tightly controls the signal transduction process triggered by ligand activation of these receptors. To identify new regulators of the endocytic trafficking of EGFR an RNA interference screen was performed for genes involved in ubiquitin conjugation and down-regulation of EGFR. The screen revealed that small interfering RNAs (siRNAs) that target the conserved ubiquitin-binding domain Uev1 increased down-regulation of EGFR. Since these siRNAs simultaneously targeted multiple genes containing a Uev1 domain, we analyzed the role of these gene products by overexpressing individual Uev1-related proteins. This analysis revealed that overexpression of Uev1A (UBE2V1) has no effect on the degradation of EGFR:EGF complexes. In contrast, overexpression of Uev1B (TMEM189-UBE2V1 isoform 2) slowed the degradation of EGF:receptor complexes. The Uev1B protein was found to strongly colocalize and associate with ubiquitin and Hrs in endosomes. Moreover, overexpression of Uev1B abrogated the ability of Hrs to colocalize with EGFR. The B-domain of Uev1B, and not the UEV-domain, was mainly responsible for the observed phenotypes suggesting the presence of a novel endosomal targeting sequence within the B-domain. Together, the data show that elevated levels of Uev1B protein in cells lead to decreased efficiency of endosomal sorting by associating with ubiquitinated proteins and Hrs.