Distinct Functions of POT1 at Telomeres

Abstract

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.Telomeres are DNA-protein complexes that protect the ends of linear eukaryotic chromosomes from illegitimate recombination, degradation, and recognition as DNA doublestrand breaks. The DNA portion of human telomeres is composed of tandem arrays of the short G-rich repetitive sequence TTAGGG in which the G-rich strand extends beyond the Crich strand, forming a 3Ј single-stranded DNA (ssDNA) overhang. Electron microscopic analysis suggests that the 3Ј overhang can loop back and invade telomeric double-stranded DNA (dsDNA) to form a large lariat structure, effectively hiding the end of the telomere (16). The protein portion of telomeres is composed of two telomeric dsDNA-binding proteins, TRF1 and TRF2 (6,25,42,46,47), and one telomeric ssDNA-binding protein, POT1 (4, 5), which form a large complex, mediated by proteins such as TIN2 and TPP1 (24,26,28,30,31,39,56,57), that maintains chromosome end stability (21,22,43,48).POT1 is a 634-amino-acid protein comprised of an N-terminal evolutionarily conserved pair of oligonucleotide/oligosaccharide (OB) folds responsible for telomeric ssDNA binding (4,5,33), with the remaining C terminus serving to bind TPP1 (31, 57) and TRF2 (55). With regard to TPP1, both recombinant (49) and purified (52) human TPP1 proteins form a heterodimer with POT1 in vitro, which enhances the association of POT1 with a G-strand telomere oligonucleotide. Moreover, in the lower eukaryote Oxytricha nova, the POT1 and TPP1 homologues TEBP␣ (4, 5) and TEBP␤ (49, 52) form a ternary complex with ssDNA (12, 23) that protects chromosome ends (15). Heterodimerization of POT1 with TPP1 thus appears to enhance POT1 function at telomeric ssDNA. With regard to TRF2, POT1 coimmunoprecipitates with TRF2 (55) and has been isolated by gel filtration in several protein subcomplexes containing TRF2 (27,30,39). POT1 thus interacts directly with telomeric ssDNA, in association with TPP1, and presumably indirectly with telomeric dsDNA, thr...

RPA and POT1

“…19 Similar to mouse cells, knockdown of POT1 in human cells also resulted in accumulation of RPA at telomeres. 20,21 Together, these studies convincingly showed that, POT1, which binds to telomeric ssDNA in a sequence-specific manner, represses ATR activation in vivo by antagonizing the non-sequence-specific binding of RPA to telomeric ssDNA.…”

“…3 Among these shelterin components, TRF1 and TRF2 interact with telomeric dsDNA in a sequence-specific manner, whereas POT1, in a complex with TPP1, binds to protection, 20,41,42 they are neither necessary nor sufficient for the localization of POT1 to telomeres. 8,20,43 Indeed, while a point mutation in the OB fold abolished POT1a binding to telomeric ssDNA in vitro, this mutant efficiently localized to telomeres in vivo. 10 These results suggest that POT1 needs to be recruited to telomeres by another mechanism to enable its direct binding to telomeric ssDNA.…”

“…8,13,[42][43][44] A POT1 mutant lacking the TPP1-binding domain failed to localize to telomeres. 8 Furthermore, a POT1 mutant unable to bind TPP1 failed to prevent RPA accumulation at telomeres, 20 suggesting that the role of POT1 in antagonizing RPA binding is dependent upon its interaction with TPP1.…”

Increased expression of telomere-related proteins correlates with resistance to radiation in human laryngeal cancer cell lines

“…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 (14)(15)(16). Among these proteins, TPP1 and POT1 directly bind single-stranded telomere DNA and interact with a number of proteins to maintain telomere length and structure (17)(18)(19). Therefore, the two proteins probably are the major players of telomere maintenance.…”

Roles of DNA Repair Proteins in Telomere Maintenance

“…The roles of human RPA in telomere maintenance Most human RPAs do not co-localize with telomerebinding protein TRF1 on the telomeres in IMR90 cells, suggesting that RPA is not a constitutive telomere binding protein. 41) However, Karlseder et al analyzed the telomere binding of RPA at various stages of the cell cycle in IMR90 by ChIP assay, and found that RPA binds to telomeres in the S phase and the G2 phase. 42) They proposed that RPA and other proteins involved in Lines represent telomereic DNA.…”