Human CST Has Independent Functions during Telomere Duplex Replication and C-Strand Fill-In

Abstract

Summary Human CST (CTC1-STN1-TEN1) is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S-phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide the first direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a novel replication factor that solves diverse replication-associated problems.

SUMOylation regulates telomere length by targeting the shelterin subunit Tpz1 Tpp1 to modulate shelterin–Stn1 interaction in fission yeast

“…An attractive possibility is that Stn1-Ten1 limits Ccq1-T93 phosphorylationdependent accumulation of telomerase to telomeres (10,26) by stimulating the recruitment of DNA polymerase α (Polα) (30,31) to reduce single-stranded DNA (ssDNA) and subsequently limit Rad3 ATR -Rad26 ATRIP kinase accumulation at telomeres. Consistently, Polα mutations cause telomere elongation in fission yeast (32), and cells lacking the telomerase inhibitors Poz1 or Rap1 exhibit a long delay in recruitment of Polα (but not the leading strand DNA polymerase e), leading to increased accumulation of ssDNA, Rad3 ATR -Rad26 ATRIP , Ccq1-T93 phosphorylation, and telomerase at telomeres (10,29).…”

Human TEN1 Maintains Telomere Integrity and Functions in Genome-wide Replication Restart

“…Telomere Length and G-overhang Analysis-Genomic DNA was isolated by proteinase K digestion and phenol chloroform extraction for G-overhang analysis or by high salt precipitation (12) for telomere length analysis, followed by overnight digestion with HinfI and MspI. For telomere length determination, restriction fragments were separated in 1% agarose by pulsefield electrophoresis (HeLa1.2.11) or standard electrophoresis (HCT116 and HT1080).…”

“…For each lane, the Exo1-resistant signal was subtracted from the untreated nondenatured signal, and the resulting G-overhang signal was normalized for loading using the signal from the denatured sample. For the cell cycle analysis of overhang length, cells were synchronized at the G 1 /S boundary with a double thymidine block as previously described (12).…”

“…Telomerase then extends the 3Ј overhang soon after duplex replication is complete (1). Finally, the complementary C-strand is filled in several hours later during late S/G 2 phase by polymerase ␣/primase (pol ␣) aided by CST (1,12,14,15).…”

CTC1‐STN1 coordinates G‐ and C‐strand synthesis to regulate telomere length

“…Defects in telomere replication due to disruption of the CST complex lead to replication fork stalling, as telomeres can adopt secondary structures that are difficult to replicate (Gu et al., 2012; Stewart et al., 2012). Stalled replication forks and the failure of stalled fork restart at telomeres initiate aberrant homologous recombination events that in part account for the catastrophic loss of total telomeric DNA observed in mouse cells devoid of CTC1 (Gu et al., 2012), or the activation of a DDR in mammalian cells (Wang et al., 2012). …”