Vertebrate POT1 restricts G-overhang length and prevents activation of a telomeric DNA damage checkpoint but is dispensable for overhang protection.

Telomeres exist as complex chromatin structures that form a protective cap for the chromosome end (43). This cap contains a number of specialized telomere proteins that regulate access to the enzymes required for telomere maintenance and prevent the chromosome terminus from being subject to DNA repair reactions such as nonhomologous end joining (NHEJ). The telomeric DNA consists of a duplex region of simple repeat sequence that terminates in a single-strand overhang on the 3′ G-rich strand. In vertebrates and other organisms with long telomeres, the telomeric DNA is folded back into a lariat-like structure in which the G overhang is tucked into the duplex region of the telomeric tract (17, 37). During DNA replication, passage of the replication fork is expected to open the T-loop structure so as to expose the overhang to telomerase and other proteins required for telomere replication (56). While the telomere is in this open configuration, the G overhang is thought to be protected by POT1 (protection of telomeres), a protein that specifically recognizes telomeric G-strand DNA (2, 56). In vertebrate cells, the double-stranded region of the telomeric DNA is bound by a core complex of six proteins: TRF1, TRF2, RAP1, TIN2, TPP1, and POT1 (10, 32, 61). TRF1 and TRF2 bind directly to the duplex T2AG3 repeats (4), while POT1 is anchored to the complex via TPP1 (21, 33, 62). Loss of TRF2 leads to a catastrophic telomere deprotection phenotype that involves removal of G overhangs by the nucleotide excision repair nuclease ERCC1/XPF followed by extensive telomere fusion via the NHEJ pathway (5, 63). Thus, TRF2 plays a key role in telomere capping. In contrast, TRF1 is more important for telomere length regulation, with overexpression leading to telomere shortening and removal leading to lengthening (50). The interplay between the components of the core TRF/RAP/TIN/TPP/POT complex in telomere length regulation appears complicated, as the length set point can be changed in unexpected ways by overexpressing wild-type and mutant versions of the various proteins (7, 21, 31, 34). In addition to the core telomere protein complex, a number of double-strand break repair proteins associate with telomeres (9). During replication of a functional telomere, the open telomere structure is transiently recognized as DNA damage and hence able to recruit proteins such as ATM and/or ATR and the MRX complex (47, 54). However, subsequent loading of specific telomere proteins somehow limits the DNA damage response and prevents activation of downstream transducers such as p53, Chk1, and Chk2. In contrast, when telomeres become dysfunctional due to critical DNA shortening or loss of TRF2, the full DNA damage response is activated, resulting in accumulation of γH2AX and other damage response proteins at the uncapped telomere (8, 18, 45) and hence formation of telomere dysfunction-induced foci (45). Telomeric G-strand binding proteins have been identified for a wide range of organisms including vertebrates, plants, fission yeast (Schizosaccharomyces pombe) (the POT1 proteins), ciliates (the telomere end binding proteins), and budding yeast (Saccharomyces cerevisiae) (Cdc13) (42, 56). All of these proteins bind telomeric G-strand DNA with high affinity and show specificity for their cognate telomeric sequence. Although they share relatively low levels of sequence identity, their DNA binding domains consist of structurally conserved oligonucleotide/oligosaccharide binding (OB) folds (20, 29, 30, 36, 49). POT1 proteins are essential for telomere protection in both fission yeast and plants. Deletion of S. pombe POT1 leads to rapid loss of telomeric and subtelomeric sequences and widespread cell death (2). Likewise, expression of a dominant negative allele of one of the Arabidopsis thaliana POT1 orthologs, Pot2, leads to extreme telomere shortening, telomere fusions, and massive genome instability (42). These telomere uncapping phenotypes, together with the highly conserved DNA binding specificity of the telomere end binding proteins and POT1 proteins, suggested that POT1 family members promote telomere protection by binding to the G-strand overhang. However, studies with human POT1 (hPOT1) did not confirm this hypothesis and revealed alternative functions. Deletion of the N-terminal OB fold resulted in an hPOT1 mutant that could not bind G-strand DNA; however, it could still associate with telomeres in vivo (34). It also caused telomere lengthening, indicating an unexpected role for hPOT1 in telomere length regulation. It is now apparent that hPOT1 binds to TPP1 through its C-terminal domain and is part of the core protein complex that coats the duplex telomeric DNA (33, 62). This finding explains why the ΔOB mutant binds to telomeres and may also explain the effects of hPOT1 on telomere length. A variety of phenotypes have been observed when hPOT1 levels are decreased by use of short-hairpin RNA (shRNA) or small interfering RNA (19, 52, 60). In general, the knockdown leads to telomere lengthening, a decrease in proliferation, and a stable 40 to 60% decrease in the amount of G-strand overhang. Surprisingly, multiple studies report only a modest (∼2-fold) increase in telomere fusions, indicating that POT1 depletion has much less effect on telomere capping than TRF2 depletion. However, one caveat of the RNA interference (RNAi) experiments is that only 70 to 95% of the POT1 was depleted; thus, the protein could have been lost preferentially from the double-stranded telomeric DNA, leaving sufficient residual protein to bind and protect the G overhangs. In light of this uncertainty, we have taken a different approach to remove POT1 from telomeres, namely, disruption of the POT1 gene in chicken DT40 cells. We used chicken DT40 cells because this avian leukosis virus-transformed B-cell line exhibits high levels of homologous recombination and hence allows efficient gene targeting (59). Contrary to early reports, DT40 cells are not p53 deficient but have functional p53 that can activate downstream targets (44). The chicken genome is similar to the human genome in that it appears to contain only one POT1 gene. Moreover, previous studies have shown that the chicken and human POT1 proteins are similar in structure, telomeric distribution, and biochemical properties (3, 35, 55). As we found that chicken POT1 is essential, we generated a conditional cell line that expresses an estrogen receptor (ER)-POT1 fusion protein (ER-POT1). This fusion protein is removed from the nucleus during growth in the absence of tamoxifen. The conditional cell line has allowed us to examine the role of POT1 in telomere protection. Our results indicate that POT1 is essential for telomere capping but that, in contrast to TRF2, POT1 is not needed to prevent G-overhang loss or telomere fusions. Rather, POT1 prevents G-overhang elongation, activation of a cell cycle checkpoint, centrosome amplification, and subsequent defects in chromosome segregation.