Telomeric 3′ Overhangs Derive from Resection by Exo1 and Apollo and Fill-In by POT1b-Associated CST

Abstract

SUMMARY A 3′ overhang is critical for the protection and maintenance of mammalian telomeres. How these overhangs are generated and whether different processing steps modify telomeres synthesized by leading- and lagging-strand DNA replication was not known. Here we evaluate changes in the telomeric overhangs through the cell cycle and at leading- and lagging-end telomeres in mouse cells lacking relevant genes. Apollo, a nuclease bound to the shelterin subunit TRF2, initiated formation of the 3′ overhang at leading-, but not lagging-end telomeres. Hyper-resection by Apollo was blocked at both ends by the shelterin protein POT1b. Exo1 extensively resected both telomere ends, generating long 3′ overhangs that transiently occurred in S/G2. CST/AAF, a DNA polymeraseα. primase accessory factor related to yeast CST, bound POT1b and shortened the extended overhangs produced by Exo1, most likely through fill-in synthesis. The results establish 3′ overhang formation as a multi-step, shelterin-controlled process that ensures functional telomeric overhangs at all chromosome ends.

Elucidation of IgH 3′ region regulatory role during class switch recombination via germline deletion

“…4) compared with wt mice. Up-regulated miRNA in 3 0 RR-deficient mice have hundreds of ARTICLE predicted targets, including Atm, Ssb1, Ino80, Exo1, Smarcd1, Dclre1b and Atmin, which have been shown to regulate DSB resection and homologous recombination [33][34][35][36][37][38] , and histone modifying enzymes Hdac9 and Esco1. Interestingly, HDAC9 is required for DSB reparation by homologous reparation 39 , and its deletion affects antibody secretion 40 .…”

Inflammatory factor receptor Toll‐like receptor 4 controls telomeres through heterochromatin protein 1 isoforms in liver cancer stem cell

“…Several studies indicate that the structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities 32. Exo1 extensively resects both ends of telomere, generating transient long 3′ overhangs in S/G2 20. Furthermore, TERRA promotes telomere shortening through exonuclease 1‐mediated resection of chromosome ends 33…”

“…Moreover, Exo1 extensively resects both telomere ends, generating transient long 3′ overhangs in S phase/G2 phase checkpoint. CST/AAF, a DNA polα primase accessory factor, binds POT1b and shortens the extended overhangs produced by Exo1, likely through fill‐in synthesis 20, 21. Furthermore, genetic variants in telomere maintenance genes are associated with genomic instability, cancer risk and cancer metastasis 22, 23, 24…”

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

“…CTC1 and STN1 were originally discovered as proteins that stimulate DNA Pol‐α: primase activity, suggesting an essential role for CST in DNA replication (Casteel et al., 2009; Miyake et al., 2009; Surovtseva et al., 2009). Targeted deletion of CTC1 in the mouse, as well as depletion of individual CST components in human cells, all results in telomere replication defects and global telomere attrition, suggesting that the CST complex is required for multiple steps of telomere replication (Feng, Hsu, Kasbek, Chaiken & Price, 2017; Gu et al., 2012; Wu, Takai & de Lange, 2012). After DNA replication, leading‐strand telomeres are initially blunt‐ended, requiring nucleolytic processing of the leading‐strand termini to generate the 3′‐overhang needed for telomerase extension of the G‐strand.…”

“…However, telomere extension is restrained by the CST complex (Chen, Redon & Lingner, 2012). The recruitment of CST to telomeres, by POT1b in mouse cells and TPP1 in human cells, in turn promotes DNA Pol‐α to perform C‐strand fill‐in reactions (Wu et al., 2012). CST: DNA Pol‐α‐mediated C‐strand fill‐in is absolutely required for telomere length maintenance, as telomerase by itself is insufficient to generate the proper duplex telomere (Feng et al., 2017; Gu et al., 2012).…”