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 ends. 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). 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 inhibitors. Here we address the mechanism by which 53BP1-RIF1-shieldin regulates the generation of recombinogenic 3' overhangs. We report that CTC1-STN1-TEN1 (CST), a complex similar to replication protein A that functions as an accessory factor of polymerase-α (Polα)-primase, 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.

We report an unexpected role for Tel2 in the expression of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Although Tel2 was identified as a budding yeast gene required for the telomere length maintenance, we found no obvious telomeric function for mammalian Tel2. Targeted gene deletion showed that mouse Tel2 is essential in embryonic development, embryonic stem (ES) cells, and embryonic fibroblasts. Conditional deletion of Tel2 from embryonic fibroblasts compromised their response to IR and UV, diminishing the activation of checkpoint kinases and their downstream effectors. The effects of Tel2 deletion correlated with significantly reduced protein levels for the PI3K-related kinases ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR), DNA-dependent protein kinase catalytic subunit ataxia (DNA-PKcs). Tel2 deletion also elicited specific depletion of the mammalian target of rapamycin (mTOR), suppressor with morphological effect on genitalia 1 (SMG1), and transformation/transcription domain-associated protein (TRRAP), and curbed mTOR signaling, indicating that Tel2 affects all six mammalian PIKKs. While Tel2 deletion did not alter PIKK mRNA levels, in vivo pulse labeling experiments showed that Tel2 controls the stability of ATM and mTOR. Each of the PIKK family members associated with Tel2 in vivo and in vitro experiments indicated that Tel2 binds to part of the HEAT repeat segments of ATM and mTOR. These data identify Tel2 as a highly conserved regulator of PIKK stability.

Human telomeres bind shelterin, the six-subunit protein complex that protects chromosome ends from the DNA damage response and regulates telomere length maintenance by telomerase. We used quantitative immunoblotting to determine the abundance and stoichiometry of the shelterin proteins in the chromatin-bound protein fraction of human cells. The abundance of shelterin components was similar in primary and transformed cells and was not correlated with telomere length. The duplex telomeric DNA binding factors in shelterin, TRF1 and TRF2, were sufficiently abundant to cover all telomeric DNA in cells with short telomeres. The TPP1⅐POT1 heterodimer was present 50 -100 copies/telomere, which is in excess of its singlestranded telomeric DNA binding sites, indicating that some of the TPP1⅐POT1 in shelterin is not associated with the singlestranded telomeric DNA. TRF2 and Rap1 were present at 1:1 stoichiometry as were TPP1 and POT1. The abundance of TIN2 was sufficient to allow each TRF1 and TRF2 to bind to TIN2. Remarkably, TPP1 and POT1 were ϳ10-fold less abundant than their TIN2 partner in shelterin, raising the question of what limits the accumulation of TPP1⅐POT1 at telomeres. Finally, we report that a 10-fold reduction in TRF2 affects the regulation of telomere length but not the protection of telomeres in tumor cell lines.Telomeres solve the two main problems associated with the organization of eukaryotic genetic information on linear chromosomes: that is, the end-replication problem and the endprotection problem. The end-replication problem is solved by the interaction of telomeres with telomerase, a telomerespecific reverse transcriptase that can replenish telomeric sequences lost during DNA replication (1). Mammalian telomeres solve the end-protection problem through their association with shelterin, a six-protein complex that specifically associates with telomeric DNA and represses both DNA damage signaling and double-strand break repair reactions at the chromosome ends (2).Shelterin binds to the duplex telomeric TTAGGG repeat array through two related DNA-binding proteins, TRF1 and TRF2 (3-5). A third DNA binding factor in shelterin, POT1, binds to single-stranded TTAGGG repeats (6). A 50 -400-nucleotide stretch of single-stranded DNA is found at the 3Ј terminus of telomeres (7) or at a telomere-internal displacement loop (D loop) that is formed by strand-invasion of the 3Ј overhang when telomeres are in the t-loop configuration (8). In vitro, POT1 can bind to TTAGGG repeats both at a 3Ј end and at the non-terminal sites found in the D loop (9 -12). The POT1 DNA binding activity is enhanced by its binding partner TPP1 (9 -12). TPP1 also connects POT1 to TIN2, which binds to TRF1 and TRF2 (13-19). The sixth component of human shelterin, Rap1, interacts with TRF2 (20).The view that the shelterin components can form a single six-subunit complex at telomeres has emerged from both the biochemical purification of the six-protein complex and studies of the protein-protein interaction network, including the finding t...

SUMMARY 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 on 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.