Schizosaccharomyces pombe protection of telomeres 1 utilizes alternate binding modes to accommodate different telomeric sequences.

Many critical cellular functions, including telomere maintenance and DNA replication, repair, and recombination, involve the management of single-stranded DNA (ssDNA) and depend on the recognition of ssDNA by OB-fold-containing proteins (1–4). While many of these interactions are not sequence specific, proteins of the telomere-end protection (TEP) family tightly and specifically bind to and protect the GT-rich 3'-ssDNA overhang present at the end of eukaryotic chromosomes (1). In the absence of this telomere cap, the free ssDNA end is subject to recognition by DNA damage machinery leading to chromosomal-end fusions, DNA resection, and chromosomal rearrangements (5–10). In addition to this critical protective function, TEP proteins interact with a suite of other telomere-associated proteins to regulate telomere homeostasis, including defining telomere structure and coordinating telomerase access to telomeric ssDNA (5, 8, 11–16). In fission yeast and humans, the TEP protein Pot1 associates with five other proteins forming a core telomere-interaction complex termed shelterin (17, 15, 18). The Tpz1 protein (TPP1 in humans) specifically interacts with Pot1 and is responsible for Pot1 recruitment to the telomere bridging Pot1 interaction with other shelterin components (19, 17). Cdc13, the budding yeast TEP protein, coordinates with a variety of other telomere-associated protein to affect telomere protection and regulation (11, 20, 21); however, to date a budding yeast shelterin complex has not been identified. While telomere structure and TEP protein interactions vary between organisms, specific recognition of telomeric ssDNA by TEP proteins is key to their function. Analysis of high-resolution structures of TEP proteins in complex with ssDNA has helped define common mechanisms of recognition, as well as those unique to each system (1). Structures of the DNA-binding domains of the TEP proteins from the yeasts Saccharyomyces cerevisiae and Schizosaccharomyces pombe, and humans reveal the common use of aromatic stacking and H-bonding networks for the specific recognition of cognate telomere sequences (22–26). Human Pot1 (hPOT1) utilizes two OB-folds (hOB1 and hOB2) to bind two repeats of the human telomere sequence, (GGTTAG)2 (27), with a binding interface characterized by four pairwise nucleotide stacks and 31 protein-ssDNA H-bonds, including 13 base-specific H-bonds located in hOB1 (25, 26). The corresponding domain in S. pombe Pot1, Pot1pN, in isolation binds a single telomere repeat (GGTTAC) (6, 28, 29) in a more compact configuration than that observed for the homologous hPOT1 OB1 (24). While the presence of the second domain in the hPOT1 structure may contribute to the differences in DNA structure, the extensive array of base-specific interactions within the Pot1pN structure indicate the compact arrangement is involved in specific recognition. The six nucleotides participate in three sets of stacking interactions, and, in addition to 15 sequence-specific protein-ssDNA H-bonds, several intraoligonucleotide H-bonds contribute to specificity. The single OB-fold of S. cerevisiae Cdc13-DBD is distinct from those of the Pot1 proteins and binds 11 nucleotides (GTGTGGGTGTG) in an extended conformation, resulting in an uncharacteristic lack of inter-nucleotide stacking (23). Additionally, the binding interface contains relatively few H-bonds; however, as S. cerevisiae telomeres do not consist of regular repeating sequence, this scarcity of H-bonding likely plays a role in accommodation of degenerate telomere sequence (30–33). These structures demonstrate the complexity of interactions contributing to recognition and highlight the adaptability of TEP OB-folds to varying specificity requirements. Pot1pN and hOB1 exhibit a high degree of structural similarity, which likely extends beyond the first OB-fold (24, 25). In the absence of any significant sequence similarity, secondary structure prediction analysis of SpPot1 supports presence of a second OB-fold (Pot1pC) (34). Biochemical studies revealed that Pot1pC has telomere-specific DNA-binding activity (35); however, a structure of Pot1pC has not been determined. The independent nature of Pot1pN and Pot1pC differs from that of hPOT1, in that hOB1 and hOB2 cannot be expressed individually (25). While Pot1pN binds a single repeat GGTTAC (6mer) (24, 29) Pot1pC minimally requires a 9mer (GGTTACGGT), representing 1.5 telomere repeats. We previously found that in addition to the composite 15mer sequence (GGTTACGGTTACGGT), Pot1-DBD, encompassing Pot1pN and Pot1pC, binds two telomere repeats, (GGTTAC)2 (12mer), with high affinity (34, 35). This ability to accommodate multiple sequence lengths contrasts with hPOT1 specificity for the precise 3'-end of the GGTTAG telomere repeat (25). The differences in Pot1 activity between homologues likely arise from the divergent nature of S. pombe and human telomeres. While the core 6-nucleotide telomere repeat sequences are highly similar to each other, differing only at position 6, S. pombe repeats are interrupted by spacer elements of variable length and sequence, yielding the general sequence GGTTAC(A/AC)0–1(G)0–7 (36, 37). To function properly at these telomeres, SpPot1 must be able to accommodate these spacers, most likely through more flexible interdomain interactions and increased malleability within the ssDNA-binding interface. Additionally, the CTD of both SpPot1 and hPOT1 contains a third putative OB-fold (38). While this domain is known to participate in protein-protein interactions (17, 19, 39), the S. pombe CTD may provide additional nucleotide contacts or allosteric effects that alter interaction with different telomeric sequences. Here we present quantitative binding studies of full-length SpPot1 and confirm that Pot1-DBD represents the complete DNA-binding domain. As 12mer and 15mer respectively represent the minimal binding and Pot1pN/Pot1pC composite sequences, we examined these two oligonucleotides for differences in binding characteristics. We find that, despite equivalent binding affinities in the low picomolar range, SpPot1 interactions with 12mer and 15mer are characterized by distinct thermodynamic and kinetic properties. Our results suggest that the molecular recognition elements in this system are exceptionally flexible and that alterations in binding mode allow for accommodation of changes in length and sequence arising from the inherent heterogeneity of S. pombe telomeres.