CST-Polα/primase, backed up by Rif1, protects telomeric ss-dsDNA junctions from damage recognition and resection

Abstract

The yeast Cdc13-Stn1-Ten1 (CST) complex is critical for telomere length regulation and telomere protection. Recent studies further implicate Pol/primase (PP) as an essential partner of CST in executing these functions, but the mechanistic bases remain poorly understood. Using the pathogenic yeast Nakaseomyces glabratus, a model amenable to mechanistic dissection of CST-PP, we analyzed the protection mechanisms of CST-PP by combining mutations that disrupt individual subunits or complex assembly with mutations in DNA damage response (DDR) and DNA repair factors. The complete loss of Cdc13 - which eliminates the CST-PP assembly - results in telomere damage recognition involving Rad9, Rad24 and Mec3, as well as aberrant resection mediated by Exo1 and Sgs1. In contrast, disrupting the interaction between CST and PP leads to proliferation defects triggered primarily by Exo1, implying a more selective protection function for PP. The involvement of the DDR clamp loader and clamp together with Exo1 points to ss-dsDNA junctions with recessed 5' ends as a crucial lesion in the absence of Cdc13. Further analysis implicates RNase H1 and H2 in promoting DNA damage, possibly by exposing such junctions. Based on similarities between Cdc13 and POT1, a telomere protein previously implicated in junctional protection, we modeled Cdc13 binding to a ss-dsDNA junction and showed that point mutations at the proposed interaction site impaired telomere protection and altered the distribution of the telomere C-strand 5' ends in vivo. We further demonstrate that Rif1, another telomere protein with junctional binding activity, supports the protection function of CST-PP. Together, our findings illuminate the molecular basis of telomere DNA damage and of CST-PP mediated telomere protection.