Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Abstract

The POT1/TEBP telomere proteins are a group of single-stranded DNA (ssDNA)-binding proteins that have long been assumed to protect the G overhang on the telomeric 3 strand. We have found that the Tetrahymena thermophila genome contains two POT1 gene homologs, POT1a and POT1b. The POT1a gene is essential, but POT1b is not. We have generated a conditional POT1a cell line and shown that POT1a depletion results in a monster cell phenotype and growth arrest. However, G-overhang structure is essentially unchanged, indicating that POT1a is not required for overhang protection. In contrast, POT1a is required for telomere length regulation. After POT1a depletion, most telomeres elongate by 400 to 500 bp, but some increase by up to 10 kb. This elongation occurs in the absence of further cell division. The growth arrest caused by POT1a depletion can be reversed by reexpression of POT1a or addition of caffeine. Thus, POT1a is required to prevent a cell cycle checkpoint that is most likely mediated by ATM or ATR (ATM and ATR are protein kinases of the PI-3 protein kinase-like family). Our findings indicate that the essential function of POT1a is to prevent a catastrophic DNA damage response. This response may be activated when nontelomeric ssDNA-binding proteins bind and protect the G overhang.In order to maintain genome integrity, the telomeric DNA from cells with linear chromosomes is packaged into a protective nucleoprotein complex (10). In the absence of this complex, the telomeres are recognized as DNA damage and subject to repair by nonhomologous end joining (33). The resulting chromosome fusions lead to genome instability (36). The protective telomeric complex is composed of a series of unique telomere proteins that bind the double-stranded region of the telomeric DNA and/or the single-strand overhang on the 3Ј G-rich strand (37). Although the exact composition of the complex varies between species, vertebrates, yeasts, plants, and ciliates all use a series of structurally related proteins to protect their telomeres. The G-overhang binding proteins all bind Gstrand DNA through a conserved OB fold motif, while the double-stranded DNA-binding proteins bind via a conserved myb motif (23,37,46).In vertebrate cells, telomeres are packaged by a core complex of six proteins which function both in telomere protection and in telomere length regulation (10). This core complex (which is sometimes called shelterin) contains two doublestranded DNA-binding proteins, TRF1 and TRF2, which anchor the complex along the length of the telomere, and the TRF1/2 interaction partners Rap1, TIN2, and TPP1. The Gstrand binding protein POT1 is also a component of the complex. Although POT1 is secured into the complex through interactions with TPP1 (17, 31, 50), POT1 molecules are also thought to bind the G-strand overhang via their OB foldcontaining DNA-binding domain (45,46). Additional telomere-associated proteins include a number of DNA damage response factors (33). However, these factors appear to bind only transiently during replication of...

How telomeric protein POT1 avoids RNA to achieve specificity for single-stranded DNA

“…Human POT1 (hPOT1) and hPOT1V2 (a splice variant of hPOT1 composed of its DNA-binding domain, used extensively to characterize POT1 structurally and biochemically) bind telomeric DNA with high affinity (K D ∼ 10 nM) and base specificity (10). POT1 is conserved among all mammals and has functional homologs in other species such as Oxytricha nova (12), Tetrahymena thermophila (13), and Schizosaccharomyces pombe (6,14). A sequence-related protein in the plant Arabidopsis thaliana associates with the telomerase ribonucleoprotein rather than the telomeric DNA (15)(16)(17).…”

Direct Single-Stranded DNA Binding by Teb1 Mediates the Recruitment of Tetrahymena thermophila Telomerase to Telomeres

“…This length of overhang is insufficient for binding both Pot1a and Teb1 (34). Because Pot1a depletion triggers runaway telomere elongation even in nondividing cells (24), telomerase exclusion from chromosome termini may depend on a low off-rate of Pot1a from bound DNA achieved in part by Pot1a interactions with other telomere proteins (25,47). Alternately, Teb1 binding to telomeres could be competed by RPA, with subsequent RPA replacement by Pot1a complexes.…”

“…This potentially constitutive assembly of holoenzyme is different from the paradigm set by yeast telomerase holoenzyme subunit regulation by the cell cycle (11,12,21,30). Nonetheless, constitutive Tetrahymena telomerase holoenzyme assembly would be consistent with the dramatic elongation of telomeres in nondividing cells depleted of the Pot1a-Tpt1-Pat1-Pat2 complex (24).…”

“…Their unusual genomic organization of a germ line micronucleus and a polyploid, fragmented-chromosome macronucleus requires tens of thousands of telomeres and an abundance of telomerase (23). In the model organism Tetrahymena, macronuclear telomeres are bound by a POT1 ortholog, Pot1a, which associates with the TPP1 ortholog Tpt1 to functionally cap telomeres and negatively regulate telomerase access (24,25). Two additional proteins, Pat1 and Pat2, interact with Pot1a-Tpt1, but their biological role is not well understood (25,26).…”

The Cech Symposium: A celebration of 25 years of ribozymes, 10 years of TERT, and 60 years of Tom

“…Carolyn Price (University of Cincinnati) showed that another of these proteins, POT-1, negatively regulates telomere length, likely through competition with telomerase for the chromosome end (Churikov et al 2006). She also concluded that, during the cell cycle, POT-1 mediates an essential checkpoint in Tetrahymena (Jacob et al 2007). Finally, Julia Cooper (Cancer Research UK) discussed how telomere-associated proteins are necessary for proper chromosome segregation during meiosis in S. pombe.…”