While the telosome forms a platform to which additional players can be recruited (Figure ), complicated interactions are also at play within the protein complex itself [11
]. TIN2 and TPP1 are critical to its assembly [65
], and the ssDNA-binding protein POT1 serves as the effector of the complex in its role of maintaining telomere integrity. Both POT1 and TPP1 contain one or more oligonucleotide/oligosaccharide-binding folds (OB folds) [66
]. Recent work has highlighted the evolutionary conservation in both structure and function among OB-fold-containing proteins participating in telomere maintenance and integrity, such as POT1 and TPP1, compared with those involved in DNA protection, such as the heterotrimeric replication protein A (RPA) complex [66
Genetic studies in yeast, Tetrahymena
, plants, humans and mice support an essential role for POT1 in maintaining telomere integrity [7
]. Unlike humans, mice contain two isoforms of POT1 - POT1a and POT1b [79
]. Recent work has shown the functional dichotomy of these two isoforms, and provided much-needed insight into the evolutionary divergence and conservation of POT1 homologs in different species [78
]. Conditional knockout studies of POT1a and POT1b suggest that both are needed for complete protection and maintenance of the telomeres [79
]. While the two proteins have overlapping functions and each may compensate to some extent for the loss of the other, they are not interchangeable. In particular, POT1a is essential for suppressing DNA damage responses at telomere termini, whereas POT1b regulates the 3' ssDNA overhangs [79
]. When the ends of chromosomes are not coated and protected by proteins, the telomeres (with or without the overhang) may be recognized as DNA damage, eliciting DNA damage response pathways. POT1b appears important for protecting the 3' overhangs from degrading nucleases. This functional difference may be achieved, in part, through the interaction of POT1a and POT1b with different sets of proteins in the telomere interactome. For example, one potential target of POT1b could be nuclease(s) that are involved in processing the 3' overhang [79
Although both TRF2 and POT1 bind telomere DNA and are required for telomere capping, recent studies indicate that they regulate distinct signaling pathways [84
]. Loss of function of TRF2 in a number of mammalian cell types (tumor and primary cell lines), and in cells from conditional TRF2-knockout mice, elicits DNA damage responses mediated mainly through the ATM pathway, whereas POT1 knockout triggers the DNA damage response pathway initiated by the protein kinase ATR (ataxia telangiectasia related) [84
]. These results are consistent with the telomere interactome map (Figure ), where TRF2 interacts with the MRN complex and DNA-PK, proteins that mediate repair of double-strand breaks, with which ATM is preferentially associated [53
]. In addition, the repression of ATR activity by POT1 is probably a result of POT1 binding telomere ssDNA and inhibiting ATR activation by blocking access of the single-strand binding protein RPA, by which ATR is recruited, to the telomere [84
]. As shown in the interactome map, few proteins are known to bind directly to POT1. How POT1 signals through pathways other than the ATR pathway merits further investigation.
In the cilate Oxytricha nova
, heterodimers of the OB-fold telomere end-binding proteins TEBP-α and TEBP-β are bound to the TTTTGGGG repeats of telomeric DNA. TEBP-α contains three OBfolds, two of which are involved in ssDNA recognition while the third interacts with TEBP-β [68
]. Human POT1 is a homolog of ciliate TEBP-α. Although TPP1 lacks an obvious OB fold, careful biochemical, structural and molecular studies have revealed that it does indeed contain an OB-fold structure, and that it is a functional homolog of the ciliate TEBP-β[66
]. Whereas TPP1 exhibits little or no telomere ssDNA-binding activities in gel-shift experiments, a POT1-TPP1-DNA ternary complex can form in these assays. TPP1 has also been shown to enhance POT1 DNA-binding activity [66
], supporting the model that POT1 may interact with DNA in the form of a heterodimer with TPP1. The TPP1-POT1 heterodimer has been postulated to modulate telomerase access to the telomeres.
Inciliates, TEBP-β canalso promote G-quadruplex formation [88
]. G-quadruplexes are tetrads of hydrogen-bonded guanine bases that can form in G-rich DNA and RNA sequences, and upon which higher-order structures can be built. Folding of telomere DNA into G-quadruplexes appears to inhibit telomerase access. This activity is unlikely to be conserved in TPP1, as TPP1 lacks the basic domain of TEBP-βthat is responsible for G-quadruplex-stimulatingactivity. In contrast, POT1 has been shown to inhibit G-quadruplex structure [89
], suggesting evolutionary divergence in G-quadruplex control mechanisms. The core telomere proteins TIN2, TRF1 and TRF2 are not found in ciliates. These proteins seem to have evolved for telomere homeostasis in vertebrates, and may provide additional mechanisms for regulating telomere G-quadruplex formation.
Both TPP1 and POT1 are critical for regulating telomere length, and POT1 is the only telomere protein identified so far that binds to telomere ssDNA. TPP1 has been shown to be able to interact with telomerase both in vitro
and in cells [66
] and its putative OB fold is required for telomerase recruitment. In addition to direct binding, the POT1-TPP1 complex appears to enhance the processivity of the telomerase component TERT in vitro
]. Consistent with this finding, expressing a TPP1 mutant lacking the OB fold resulted in modest telomere shortening in human cells compared to parental cells or cells expressing full-length TPP1 [66
]. Because TPP1 on its own does not bind ssDNA, this probably means that POT1 and TPP1 function together to recruit telomerase to telomeric ssDNA through the TPP1 OB fold, in addition to protecting telomere ends and negatively regulating telomerase access. Generally, telomeres only become accessible to the telomerase during the S phase of the cell cycle. This is achieved through multiple mechanisms, including regulation of telomerase expression and activity, sequestering of telomeres, and coating of telomeres with telomere-binding proteins such as POT1, which presumably serves to block telomerase access.
The realization that there are two classes of OB-fold-containing proteins with distinct functions has in turn helped to establish a unified model regarding the function of OB-fold-containing proteins in telomere overhang binding (Table ) [66
]. While much conservation exists between the various OB-fold-containing complexes, differences such as DNA-binding specificities, domain structures, and interaction partners help to set these proteins apart. From yeast to human, RPA-like or TEBP heteromultimeric complexes may have evolved for the more specialized function of ssDNA protection at the telomeres [66
Evolution of telomere-end-binding proteins containing the OB fold