53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polα-dependent fill-in.

In DNA repair, the resection of double-strand breaks dictates the choice between homology-directed repair-which requires a 3' overhang-and classical non-homologous end joining, which can join unresected ends1,2. BRCA1-mutant cancers show minimal resection of double-strand breaks, which renders them deficient in homology-directed repair and sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1)3-8. When BRCA1 is absent, the resection of double-strand breaks is thought to be prevented by 53BP1, RIF1 and the REV7-SHLD1-SHLD2-SHLD3 (shieldin) complex, and loss of these factors diminishes sensitivity to PARP1 inhibitors4,6-9. Here we address the mechanism by which 53BP1-RIF1-shieldin regulates the generation of recombinogenic 3' overhangs. We report that CTC1-STN1-TEN1 (CST)10, a complex similar to replication protein A that functions as an accessory factor of polymerase-α (Polα)-primase11, is a downstream effector in the 53BP1 pathway. CST interacts with shieldin and localizes with Polα to sites of DNA damage in a 53BP1- and shieldin-dependent manner. As with loss of 53BP1, RIF1 or shieldin, the depletion of CST leads to increased resection. In BRCA1-deficient cells, CST blocks RAD51 loading and promotes the efficacy of PARP1 inhibitors. In addition, Polα inhibition diminishes the effect of PARP1 inhibitors. These data suggest that CST-Polα-mediated fill-in helps to control the repair of double-strand breaks by 53BP1, RIF1 and shieldin.

TPP1 Blocks an ATR-Mediated Resection Mechanism at Telomeres.

The regulation of 5' end resection at DSBs and telomeres prevents genome instability. DSB resection is positively and negatively regulated by ATM signaling through CtIP/MRN and 53BP1-bound Rif1, respectively. Similarly, telomeres lacking TRF2 undergo ATM-controlled CtIP-dependent hyper-resection when the repression by 53BP1/Rif1 is alleviated. However, telomere resection in the absence of 53BP1/Rif1 is more extensive upon complete removal of shelterin, indicating additional protection against resection by shelterin. Here we show that TPP1 and POT1a/b in shelterin block a resection pathway distinct from that repressed by TRF2. This second pathway is regulated by ATR signaling, involves Exo1 and BLM, and is inhibited by 53BP1/Rif1. Thus, mammalian cells have two distinct 5' end-resection pathways that are regulated by DNA damage signaling, in part through Rif1-mediated inhibition. The data show that telomeres are protected from hyper-resection through the repression of the ATM and ATR kinases by TRF2 and TPP1-bound POT1a/b, respectively.

TRF1 negotiates TTAGGG repeat-associated replication problems by recruiting the BLM helicase and the TPP1/POT1 repressor of ATR signaling.

The semiconservative replication of telomeres is facilitated by the shelterin component TRF1. Without TRF1, replication forks stall in the telomeric repeats, leading to ATR kinase signaling upon S-phase progression, fragile metaphase telomeres that resemble the common fragile sites (CFSs), and the association of sister telomeres. In contrast, TRF1 does not contribute significantly to the end protection functions of shelterin. We addressed the mechanism of TRF1 action using mouse conditional knockouts of BLM, TRF1, TPP1, and Rap1 in combination with expression of TRF1 and TIN2 mutants. The data establish that TRF1 binds BLM to facilitate lagging but not leading strand telomeric DNA synthesis. As the template for lagging strand telomeric DNA synthesis is the TTAGGG repeat strand, TRF1-bound BLM is likely required to remove secondary structures formed by these sequences. In addition, the data establish that TRF1 deploys TIN2 and the TPP1/POT1 heterodimers in shelterin to prevent ATR during telomere replication and repress the accompanying sister telomere associations. Thus, TRF1 uses two distinct mechanisms to promote replication of telomeric DNA and circumvent the consequences of replication stress. These data are relevant to the expression of CFSs and provide insights into TIN2, which is compromised in dyskeratosis congenita (DC) and related disorders.

Telomere protection by TPP1/POT1 requires tethering to TIN2.

To prevent ATR activation, telomeres deploy the single-stranded DNA binding activity of TPP1/POT1a. POT1a blocks the binding of RPA to telomeres, suggesting that ATR is repressed through RPA exclusion. However, comparison of the DNA binding affinities and abundance of TPP1/POT1a and RPA indicates that TPP1/POT1a by itself is unlikely to exclude RPA. We therefore analyzed the central shelterin protein TIN2, which links TPP1/POT1a (and POT1b) to TRF1 and TRF2 on the double-stranded telomeric DNA. Upon TIN2 deletion, telomeres lost TPP1/POT1a, accumulated RPA, elicited an ATR signal, and showed all other phenotypes of POT1a/b deletion. TIN2 also affected the TRF2-dependent repression of ATM kinase signaling but not to TRF2-mediated inhibition of telomere fusions. Thus, while TIN2 has a minor contribution to the repression of ATM by TRF2, its major role is to stabilize TPP1/POT1a on the ss telomeric DNA, thereby allowing effective exclusion of RPA and repression of ATR signaling.

Fission yeast Pot1 and RecQ helicase are required for efficient chromosome segregation.

Pot1 is a single-stranded telomere-binding protein that is conserved from fission yeast to mammals. Deletion of Schizosaccharomyces pombe pot1(+) causes immediate telomere loss. S. pombe Rqh1 is a homolog of the human RecQ helicase WRN, which plays essential roles in the maintenance of genomic stability. Here, we demonstrate that a pot1Δ rqh1-hd (helicase-dead) double mutant maintains telomeres that are dependent on Rad51-mediated homologous recombination. Interestingly, the pot1Δ rqh1-hd double mutant displays a "cut" (cell untimely torn) phenotype and is sensitive to the antimicrotubule drug thiabendazole (TBZ). Moreover, the chromosome ends of the double mutant do not enter the pulsed-field electrophoresis gel. These results suggest that the entangled chromosome ends in the pot1Δ rqh1-hd double mutant inhibit chromosome segregation, signifying that Pot1 and Rqh1 are required for efficient chromosome segregation. We also found that POT1 knockdown, WRN-deficient human cells are sensitive to the antimicrotubule drug vinblastine, implying that some of the functions of S. pombe Pot1 and Rqh1 may be conserved in their respective human counterparts POT1 and WRN.

Expression of mutant RPA in human cancer cells causes telomere shortening.

Replication protein A (RPA) binds to single-stranded DNA generated during DNA replication and other processes. The roles of RPA in telomere maintenance have been demonstrated in yeasts, but not in telomerase-positive human cells. In this study, we found that expression of mutant RPA70 in human cells caused telomere shortening, suggesting that RPA is required for telomere-length regulation in human cancer cells.

Telomere protection by TPP1 is mediated by POT1a and POT1b.

Mammalian telomeres are protected by the shelterin complex, which contains single-stranded telomeric DNA binding proteins (POT1a and POT1b in rodents, POT1 in other mammals). Mouse POT1a prevents the activation of the ATR kinase and contributes to the repression of the nonhomologous end-joining pathway (NHEJ) at newly replicated telomeres. POT1b represses unscheduled resection of the 5'-ended telomeric DNA strand, resulting in long 3' overhangs in POT1b KO cells. Both POT1 proteins bind TPP1, forming heterodimers that bind to other proteins in shelterin. Short hairpin RNA (shRNA)-mediated depletion had previously demonstrated that TPP1 contributes to the normal function of POT1a and POT1b. However, these experiments did not establish whether TPP1 has additional functions in shelterin. Here we report on the phenotypes of the conditional deletion of TPP1 from mouse embryo fibroblasts. TPP1 deletion resulted in the release of POT1a and POT1b from chromatin and loss of these proteins from telomeres, indicating that TPP1 is required for the telomere association of POT1a and POT1b but not for their stability. The telomere dysfunction phenotypes associated with deletion of TPP1 were identical to those of POT1a/POT1b DKO cells. No additional telomere dysfunction phenotypes were observed, establishing that the main role of TPP1 is to allow POT1a and POT1b to protect chromosome ends.

Functional dissection of human and mouse POT1 proteins.

The single-stranded telomeric DNA binding protein POT1 protects mammalian chromosome ends from the ATR-dependent DNA damage response, regulates telomerase-mediated telomere extension, and limits 5'-end resection at telomere termini. Whereas most mammals have a single POT1 gene, mice have two POT1 proteins that are functionally distinct. POT1a represses the DNA damage response, and POT1b controls 5'-end resection. In contrast, as we report here, POT1a and POT1b do not differ in their ability to repress telomere recombination. By swapping domains, we show that the DNA binding domain of POT1a specifies its ability to repress the DNA damage response. However, no differences were detected in the in vitro DNA binding features of POT1a and POT1b. In contrast to the repression of ATR signaling by POT1a, the ability of POT1b to control 5'-end resection was found to require two regions in the C terminus, one corresponding to the TPP1 binding domain and a second representing a new domain located between amino acids (aa) 300 and 350. Interestingly, the DNA binding domain of human POT1 can replace that of POT1a to repress ATR signaling, and the POT1b region from aa 300 to 350 required for the regulation of the telomere terminus is functionally conserved in human POT1. Thus, human POT1 combines the features of POT1a and POT1b.

Fission yeast Taz1 and RPA are synergistically required to prevent rapid telomere loss.

The telomere complex must allow nucleases and helicases to process chromosome ends to make them substrates for telomerase, while preventing these same activities from disrupting chromosome end-protection. Replication protein A (RPA) binds to single-stranded DNA and is required for DNA replication, recombination, repair, and telomere maintenance. In fission yeast, the telomere binding protein Taz1 protects telomeres and negatively regulates telomerase. Here, we show that taz1-d rad11-D223Y double mutants lose their telomeric DNA, indicating that RPA (Rad11) and Taz1 are synergistically required to prevent telomere loss. Telomere loss in the taz1-d rad11-D223Y double mutants was suppressed by additional mutation of the helicase domain in a RecQ helicase (Rqh1), or by overexpression of Pot1, a single-strand telomere binding protein that is essential for protection of chromosome ends. From our results, we propose that in the absence of Taz1 and functional RPA, Pot1 cannot function properly and the helicase activity of Rqh1 promotes telomere loss. Our results suggest that controlling the activity of Rqh1 at telomeres is critical for the prevention of genomic instability.

Fission yeast Dna2 is required for generation of the telomeric single-strand overhang.

It has been suggested that the Schizosaccharomyces pombe Rad50 (Rad50-Rad32-Nbs1) complex is required for the resection of the C-rich strand at telomere ends in taz1-d cells. However, the nuclease-deficient Rad32-D25A mutant can still resect the C-rich strand, suggesting the existence of a nuclease that resects the C-rich strand. Here, we demonstrate that a taz1-d dna2-2C double mutant lost the G-rich overhang at a semipermissive temperature. The amount of G-rich overhang in S phase in the dna2-C2 mutant was lower than that in wild-type cells at the semipermissive temperature. Dna2 bound to telomere DNA in a chromatin immunoprecipitation assay. Moreover, telomere length decreased with each generation after shift of the dna2-2C mutant to the semipermissive temperature. These results suggest that Dna2 is involved in the generation of G-rich overhangs in both wild-type cells and taz1-d cells. The dna2-C2 mutant was not gamma ray sensitive at the semipermissive temperature, suggesting that the ability to process double-strand break (DSB) ends was not affected in the dna2-C2 mutant. Our results reveal that DSB ends and telomere ends are processed by different mechanisms.

Fission yeast Arp6 is required for telomere silencing, but functions independently of Swi6.

The actin-related proteins (Arps), which are subdivided into at least eight subfamilies, are conserved from yeast to humans. A member of the Arp6 subfamily in Drosophila, Arp4/Arp6, co-localizes with heterochromatin protein 1 (HP1) in pericentric heterochromatin. Fission yeast Schizosaccharomyces pombe possesses both an HP1 homolog and an Arp6 homolog. However, the function of S.pombe Arp6 has not been characterized yet. We found that deletion of arp6(+) impaired telomere silencing, but did not affect centromere silencing. Chromatin immunoprecipitation assays revealed that Arp6 bound to the telomere region. However, unlike Drosophila Arp4/Arp6, S.pombe Arp6 was distributed throughout nuclei. The binding of Arp6 to telomere DNA was not affected by deletion of swi6(+). Moreover, the binding of Swi6 to telomere ends was not affected by deletion of arp6(+). These results suggest that Arp6 and Swi6 function independently at telomere ends. We propose that the Arp6-mediated repression mechanism works side by side with Swi6-based telomere silencing in S.pombe.

Fission yeast Rhp51 is required for the maintenance of telomere structure in the absence of the Ku heterodimer.

The Schizosaccharomyces pombe Ku70-Ku80 heterodimer is required for telomere length regulation. Lack of pku70+ results in telomere shortening and striking rearrangements of telomere-associated sequences. We found that the rearrangements of telomere-associated sequences in pku80+ mutants are Rhp51 dependent, but not Rad50 dependent. Rhp51 bound to telomere ends when the Ku heterodimer was not present at telomere ends. We also found that the single-stranded G-rich tails increased in S phase in wild-type strains, while deletion of pku70+ increased the single-stranded overhang in both G2 and S phase. Based on these observations, we propose that Rhp51 binds to the G-rich overhang and promotes homologous pairing between two different telomere ends in the absence of Ku heterodimer. Moreover, pku80 rhp51 double mutants showed a significantly reduced telomere hybridization signal. Our results suggest that, although Ku heterodimer sequesters Rhp51 from telomere ends to inhibit homologous recombination activity, Rhp51 plays important roles for the maintenance of telomere ends in the absence of the Ku heterodimer.