CST does not evict elongating telomerase but prevents initiation by ssDNA binding

Abstract

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

Structure of human CST-pol-α/primase bound to a telomeric overhang poised for initiation of telomere C-strand synthesis

“…Both of these protein complexes share functions 5,6 and structural homology 7 . At mammalian telomeres, CST inhibits extension of telomere G-rich overhangs 8,9 and facilitates pol-α/primase to synthesize the complementary C-strand. The latter process is known as the telomere C-strand fill-in 10,11 .…”

Reconstitution of a telomeric replicon organized by CST

“…When we express recombinant human CST in HEK-293T cells, the affinity-purified CST also contains the four subunits of Polα-primase 11 . Because Polα-primase is not overexpressed, it represents the naturally occurring enzyme.…”

“…If telomeric repeats indeed define origins of priming and replication due to the DNA-binding specificity of CST, then C-strand synthesis should require the DNA-binding activity of CST. We tested this hypothesis with two DNA-binding mutants of the CTC1 subunit of CST, both of which still bind Polα-primase 11 . Each mutant has groups ('g') of two or three amino acids mutated.…”

“…Combining telomerase, a short telomeric ssDNA primer, and CST-Polα-primase gives complete telomeric DNA replication, resulting in the same sort of ssDNA 3'-overhang found naturally on human telomeres. We conclude that the CST complex not only terminates telomerase extension 10,11 and recruits Polα-primase to telomeric ssDNA 4,12,13 , but it also orchestrates C-strand synthesis. Because replication of the telomere has features distinct from replication of the rest of the genome, targeting telomere-replication components including CST holds promise for cancer therapeutics.…”

“…In these experiments, synthetic oligonucleotides with telomere-like sequences serve as primers for telomerase. Human telomerase extension terminates with the help of CST, which sequesters free primers and prevents re-initiation 10,11 .…”