Abstract
Comprised of telomeric TTAGGG repeats and shelterin, telomeres ensure that the natural ends of chromosomes remain impervious to the DNA damage response. Telomeres carry a long constitutive 3' overhang that can bind replication protein A (RPA) and activate the ATR Ser/Thr kinase (ATR), which induces cell cycle arrest. A single-stranded (ss) TTAGGG repeat-binding protein in mouse shelterin, POT1a, has been proposed to repress ATR signaling by preventing RPA binding. Repression of ATR at telomeres requires tethering of POT1a to the other shelterin subunits situated on the double-stranded (ds) telomeric DNA. The simplest model of ATR repression, the "tethered exclusion model," suggests that the only critical features of POT1a are its connection to shelterin and its binding to ss telomeric DNA. In agreement with the model, we show here that a shelterin-tethered variant of RPA70 (lacking the ATR recruitment domain) can repress ATR signaling at telomeres that lack POT1a. However, arguing against the tethered exclusion model, the nearly identical POT1b subunit of shelterin has been shown to be much less proficient than POT1a in repression of ATR. We now show that POT1b has the intrinsic ability to fully repress ATR but is prevented from doing so when bound to Ctc1, Stn1, Ten1 (CST), the complex needed for telomere end processing. These results establish that shelterin represses ATR with a tethered ssDNA-binding domain that excludes RPA from the 3' overhang and also reveal an unexpected effect of CST on the ability of POT1b to repress ATR.
Highlights
One of the many tasks of the telomeric shelterin complex is to repress the ATR-dependent DNA damage response at the natural ends of chromosomes
This part of RPA70 was fused to the C terminus of POT1a, which is the part of POT1a that interacts with TPP1 and ensures association with shelterin
The shelterin-tethered chimera of RPA70 and the C terminus of POT1a (⌬70-POTaC) is remarkably proficient in repressing ATR signaling at telomeres, not as effective as WT POT1a
Summary
Comprised of telomeric TTAGGG repeats and shelterin, telomeres ensure that the natural ends of chromosomes remain impervious to the DNA damage response. Telomeres lacking POT1 proteins accumulate RPA, which is not detectable at functional telomeres [11, 26] This observation suggested a simple competition model whereby the binding of POT1 to the ssDNA prevents RPA-dependent ATR activation (9, 11, 26 –28). In its simplest form, the RPA exclusion model predicts that any shelterin-tethered protein with the ability to bind ss TTAGGG repeats can repress ATR signaling at telomeres. This prediction is not met in the context of POT1b, which can repress ATR signaling in G1 but not in S phase. The results provide evidence for the idea that ATR repression involves RPA exclusion by a shelterin-tethered ssDNA-binding protein with no other specialized features
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