The yeast CST and Polα/primase complexes act in concert to ensure proper telomere maintenance and protection

Polα/primase (PP), the polymerase that initiates DNA synthesis at replication origins, also completes the task of genome duplication by synthesizing the telomere C-strand under the control of the CTC1/CDC13-STN1-TEN1 (CST) complex. Using cryo-electron microscopy (cryo-EM) structures of the human CST-Polα/primase-DNA complex as guides in conjunction with AlphaFold modeling, we identified structural elements in yeast CST and PP that promote complex formation. Mutating these structures in Candida glabrata Stn1, Ten1, Pri1, and Pri2 abrogated the stimulatory activity of CST on PP in vitro, supporting the functional relevance of the physical contacts in cryo-EM structures as well as the conservation of mechanisms between yeast and humans. Introducing these mutations into C. glabrata yielded two distinct groups of mutants. One group exhibited progressive, telomerase-dependent telomere elongation without evidence of DNA damage. The other manifested slow growth, telomere length heterogeneity, single-stranded DNA accumulation and elevated C-circles, which are indicative of telomere deprotection. These telomere deprotection phenotypes are altered or suppressed by mutations in multiple DNA damage response (DDR) and DNA repair factors. We conclude that in yeast, the telomerase inhibition and telomere protection function previously ascribed to the CST complex are mediated jointly by both CST and Polα/primase, highlighting the critical importance of a replicative DNA polymerase in telomere regulation.