Abstract
Telomeres carry a constitutive 3′ overhang that can bind RPA and activate ATR signaling. POT1a, a single-stranded (ss) DNA binding protein in mouse shelterin, has been proposed to repress ATR signaling by preventing RPA binding. Repression of ATR at telomeres requires the TPP1/TIN2 mediated tethering of POT1a to the the rest of the shelterin complex situated on the ds telomeric DNA. The simplest version of the tethered exclusion model for ATR repression suggests that the only critical features of POT1a are its connection to shelterin and its binding to ss telomeric DNA binding. In agreement with the model, we show that a shelterin-tethered RPA70 mutant, lacking the ATR recruitment domain, is effective in repressing ATR signaling at telomeres. However, arguing against the simple tethered exclusion model, the nearly identical POT1b subunit of shelterin is much less proficient in ATR repression than POT1a. We now show that POT1b has the intrinsic ability to fully repress ATR but is prevented from doing so when bound to the CST/Polα/primase complex. The data establish that shelterin represses ATR with a tethered ssDNA-binding domain that excludes RPA from the overhang and suggest that ATR repression does not require the interaction of POT1 with the 3′ end or G4 DNA.
