Added by telomerase, arrays of TTAGGG repeats specify the ends of human chromosomes. A complex formed by six telomere-specific proteins associates with this sequence and protects chromosome ends. By analogy to other chromosomal protein complexes such as condensin and cohesin, I will refer to this complex as shelterin. Three shelterin subunits, TRF1, TRF2, and POT1 directly recognize TTAGGG repeats. They are interconnected by three additional shelterin proteins, TIN2, TPP1, and Rap1, forming a complex that allows cells to distinguish telomeres from sites of DNA damage. Without the protective activity of shelterin, telomeres are no longer hidden from the DNA damage surveillance and chromosome ends are inappropriately processed by DNA repair pathways. How does shelterin avert these events? The current data argue that shelterin is not a static structural component of the telomere. Instead, shelterin is emerging as a protein complex with DNA remodeling activity that acts together with several associated DNA repair factors to change the structure of the telomeric DNA, thereby protecting chromosome ends. Six shelterin subunits: TRF1, TRF2, TIN2, Rap1, TPP1, and POT1.

/pdf/shelterin-the-protein-complex-that-shapes-and-safeguards-4nn8ijxqxb.pdf

Shelterin: the protein complex that shapes and safeguards human telomeres

The genomes of prokaryotes and eukaryotic organelles are usually circular as are most plasmids and viral genomes. In contrast, the nuclear genomes of eukaryotes are organized on linear chromosomes, which require mechanisms to protect and replicate DNA ends. Eukaryotes navigate these problems with the advent of telomeres, protective nucleoprotein complexes at the ends of linear chromosomes, and telomerase, the enzyme that maintains the DNA in these structures. Mammalian telomeres contain a specific protein complex, shelterin, that functions to protect chromosome ends from all aspects of the DNA damage response and regulates telomere maintenance by telomerase. Recent experiments, discussed here, have revealed how shelterin represses the ATM and ATR kinase signaling pathways and hides chromosome ends from nonhomologous end joining and homology-directed repair.

http://delangelab.rockefeller.edu/pubs/108_Palm_AnnRevGen2008.pdf

How Shelterin Protects Mammalian Telomeres

CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.

/pdf/repair-of-double-strand-breaks-induced-by-crispr-cas9-leads-3pklmwoxno.pdf

Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

The development of new CRISPR-Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR-Cas-derived genome editing agents-nucleases, base editors, transposases/recombinases and prime editors-are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics.

Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors

The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.

SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin

DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks

http://delangelab.rockefeller.edu/pubs/84_Ye_JBC_2004.pdf

TIN2 Binds TRF1 and TRF2 Simultaneously and Stabilizes the TRF2 Complex on Telomeres

Mammalian telomeres are protected by a six-protein complex: shelterin. Shelterin contains two closely related proteins (TRF1 and TRF2), which recruit various proteins to telomeres. We dissect the interactions of TRF1 and TRF2 with their shared binding partner (TIN2) and other shelterin accessory factors. TRF1 recognizes TIN2 using a conserved molecular surface in its TRF homology (TRFH) domain. However, this same surface does not act as a TIN2 binding site in TRF2, and TIN2 binding to TRF2 is mediated by a region outside the TRFH domain. Instead, the TRFH docking site of TRF2 binds a shelterin accessory factor (Apollo), which does not interact with the TRFH domain of TRF1. Conversely, the TRFH domain of TRF1, but not of TRF2, interacts with another shelterin-associated factor: PinX1.

https://science.sciencemag.org/content/sci/319/5866/1092.full.pdf

A shared docking motif in TRF1 and TRF2 used for differential recruitment of telomeric proteins.

The ends of linear chromosomes suffer two problems: They cannot be replicated to their termini, resulting in loss of terminal sequences; and they can be mistakenly sensed as DNA double-strand breaks, activating DNA repair pathways that can result in serious genome derangement. These problems are solved by the addition of telomeres, repeat sequences at the ends of chromosomes, which are shielded by a protein complex called shelterin. Sfeir et al. (p. [1657][1]) show that the mouse Rap1 protein, which is part of the shelterin complex and which binds to a second shelterin protein called TRF2, helps prevent telomeres undergoing unscheduled homologous recombination. Such recombination could threaten telomere integrity by generating sequence exchanges between sister telomeres resulting in critically shortened telomeres.

 [1]: /lookup/volpage/327/1657

/pdf/loss-of-rap1-induces-telomere-recombination-in-the-absence-4wg3h1396g.pdf

Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal.

https://delangelab.rockefeller.edu/pubs/94_van_Overbeek_Curret_Biology_2006sup.pdf

Megan van Overbeek

Papers

DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks

TIN2 Binds TRF1 and TRF2 Simultaneously and Stabilizes the TRF2 Complex on Telomeres

A shared docking motif in TRF1 and TRF2 used for differential recruitment of telomeric proteins.

Loss of Rap1 induces telomere recombination in the absence of NHEJ or a DNA damage signal.

Apollo, an artemis-related nuclease, interacts with TRF2 and protects human telomeres in S phase