The human CST complex is a terminator of telomerase activity

The lengths of human telomeres, which protect chromosome ends from degradation and end fusions(1,2), are crucial determinants of cell lifespan(3). During embryogenesis and in cancer, the telomerase enzyme counteracts telomeric DNA shortening. As shown in cancer cells, human telomerase binds the shelterin component TPP1 at telomeres(4,5) during the S phase of the cell cycle, and adds similar to 60 nucleotides in a single round of extension(6), after which telomerase is turned off by unknown mechanisms. Here we show that the human CST (CTC1, STN1 and TEN1) complex, previously implicated in telomere protection and DNA metabolism(7-11), inhibits telomerase activity through primer sequestration and physical interaction with the protection of telomeres 1 (POT1)-TPP1 telomerase processivity factor(12,13). CST competes with POT1-TPP1 for telomeric DNA, and CST-telomeric-DNA binding increases during late S/G2 phase only on telomerase action, coinciding with telomerase shut-off. Depletion of CST allows excessive telomerase activity, promoting telomere elongation. We propose that through binding of the telomerase-extended telomere, CST limits telomerase action at individual telomeres to approximately one binding and extension event per cell cycle. Our findings define the sequence of events that occur to first enable and then terminate telomerase-mediated telomere elongation.

The double life of UPF1 in RNA and DNA stability pathways

Joachim Lingner

The DNA and RNA helicase UPF1 is well known for its central role in Nonsense Mediated RNA Decay (NMD), which promotes degradation of mRNAs containing premature stop codons. However, we have recently demonstrated that human UPF1 is also essential for DNA replication and S phase progression. This function appears to be independent of NMD, which is not required for cell cycle progression. UPF1 physically interacts with the replicative DNA polymerase delta and it associates with chromatin during S phase and upon DNA damage in an ATR-dependent manner. Intriguingly, the human NMD-kinase SMG1 is also involved in genome stability pathways and the human NMD-factor EST1A/SMG6 is telomerase-associated and has been implicated in telomere maintenance. Here we review the recent findings, which uncovered the direct roles of UPF1 and other NMD-factors in DNA replication and genome maintenance pathways and suggest functional connections between RNA and DNA metabolism.

2006

CST for the grand finale of telomere replication

Liuh-Yow Chen, Joachim Lingner

Telomeric DNA at eukaryotic chromosome ends terminates with single stranded 3' G-rich overhangs. The overhang is generated by the interplay of several dynamic processes including semiconservative DNA replication, 3' end elongation by telomerase, C-strand fill-in synthesis and nucleolytic processing. The mammalian CST (CTC1-STN1-TEN1) complex is directly involved at several stages of telomere end formation. Elucidation of its structural organization and identification of interaction partners support the notion that mammalian CST is, as its yeast counterpart, a RPA-like complex. CST binding at mammalian telomere 3' overhangs increases upon their elongation by telomerase. Formation of a trimeric CST complex at telomeric 3'overhangs leads to telomerase inhibition and at the same time mediates a physical interaction with DNA polymerase-α. Thus CST seems to play critical roles in coordinating telomerase elongation and fill-in synthesis to complete telomere replication.

Landes Bioscience2013

A Quantitative Telomeric Chromatin Isolation Protocol (Q-TIP) to Characterize Telomere Composition Changes

Larissa Grolimund

Telomeres protect linear ends of eukaryotic chromosomes by preventing chromosomal end-to-end fusions and telomere attrition. They consist of repetitive DNA sequences, telomeric repeat containing RNAs (TERRAs) and proteins. Telomeres play a crucial role in chromosome stability, cancer biology, and ageing. With each cell division, telomeres shorten, as the replication machinery of the cell is not capable to fully copy the very terminal part of chromosomes. Therefore, after a certain number of cell divisions, telomeres get critically short and elicit signals in order to stop the proliferation. A cell can circumvent the signal for critically short telomeres by activating mechanisms that elongate telomeres. In most of the cases, telomere elongation is achieved by the expression of the telomerase enzyme. In this case, cells gain the ability to divide indefinitely, which can, in combination with other events, eventually lead to cancer. The telomeric protein composition changes during tumorigenesis, aging and in telomere syndromes are poorly defined. Here, we develop a quantitative telomeric chromatin isolation protocol (q-TIP) for human cells in which chromatin is crosslinked, immunopurified and analyzed by mass spectrometry. Q-TIP involves stable isotope labeling by amino acids in cell culture (SILAC) in order to compare and identify quantitative differences in telomere protein composition of cells from various states. With q-TIP, we specifically enrich telomeric DNA and all shelterin components. We identify and validate known and novel telomere associated polypeptides including all THO subunits, SMCHD1 and LRIF1. We apply q-TIP to long and short telomeres and detect increased density of SMCHD1 and LRIF1 and increased association of the shelterin components TRF1, TIN2, TPP1 and POT1 with long telomeres. Our results validate q-TIP to study telomeric states during normal development and in disease.

EPFL2013

A Quantitative Telomeric Chromatin Isolation Protocol (Q-TIP) to Characterize Telomere Composition Changes

Larissa Grolimund

EPFL2013