Reconstitution of a telomeric replicon organized by CST

Abstract

Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins1. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells2. This replication requires telomerase3 extension of the single-stranded DNA (ssDNA) of the telomeric G-strand ((TTAGGG)n); the synthesis of the complementary C-strand ((CCCTAA)n) is much less well characterized. The CST (CTC1–STN1–TEN1) protein complex, a DNA polymerase α-primase accessory factor4,5, is known to be required for telomere replication in vivo6–9, and the molecular analysis presented here reveals key features of its mechanism. We find that human CST uses its ssDNA-binding activity to specify the origins for telomeric C-strand synthesis by bound Polα-primase. CST-organized DNA polymerization can copy a telomeric DNA template that folds into G-quadruplex structures, but the challenges presented by this template probably contribute to telomere replication problems observed in vivo. 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 extension10,11 and recruits Polα–primase to telomeric ssDNA4,12,13 but 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.

Lagging Strand Initiation Processes in DNA Replication of Eukaryotes – Strings of Highly Coordinated Reactions Governed by Multiprotein Complexes

“…Recently, three eukaryotic model systems have shed light on this central process: The lagging strand synthesis of telomere sequences using Pol-prim and CTC1-STN1-TEN1 (CST) complex (Figure 2; N.B in mouse CTC1 and STN1 are equivalent to the AAF132 and AAF44 subunits of alpha-Accessory factor (AAF) [48]). The SV40 system using Tag, RPA plus Pol-prim, and the cellular replication system with purified yeast and human proteins have advanced our understanding [17,18,25,29,32,46,[49][50][51][52][53][54][55][56][57][58].…”

CST/Polα/primase-mediated fill-in synthesis at DSBs

“…CST/Polα/primase has diverse roles in genome maintenance, including its critical role in maintaining the C-rich telomeric repeat strand at chromosome ends [ 29 , 34–40 ], resolving G4 structures that hinder replication at telomeres and other sites in the genome [ 26 , 29–31 , 57 ], and was proposed to mediate fill-in synthesis at DSBs [ 8 ] (see Box 2 and Figure 2 ). Data published in the past year now provide strong support for CST/Polα/primase-mediated fill-in synthesis at resected DSBs.…”

Telomeres and Their Neighbors

“…Two major telomere protecting complexes have been described, CST and shelterin. These were initially thought to be alternative mutually exclusive systems, but the search for homologues revealed that many eukaryotes, including humans, had both systems able to work in parallel [ 275 , 276 , 277 , 278 ]. Continuing research focused on looking for homologues of human systems across all eukaryotes, however this approach has had only partial success (reviewed in [ 279 ]).…”

“…The CST complex is largely conserved in eukaryotes [ 280 ] in terms of function, if not necessarily the sequence of its components [ 281 , 282 , 283 ]. CST binds ssDNA and recruits Pol1α primase for C-rich strand synthesis and also has a role in preventing stalled replication forks (for recent advancements see [ 278 ] and references herein). In comparison, shelterin (reviewed in [ 272 ]) coats telomeric DNA generally and interacts with telomerase for G-rich strand synthesis.…”

“…TRF2 alone can also recruit repressor/activator protein 1 (RAP1) as the sixth member of core shelterin and the interactions between shelterin subunits can generally occur across multiple protein surfaces [ 291 ]. TPP1 in complex with POT1 interacts with telomerase as part of the coordination of telomerase and shelterin protein complexes [ 278 , 292 ].…”