The human telomere and its relationship to human disease, therapy, and tissue engineering

Ian K. Moon, Michael B. Jarstfer

University of North Carolina, School of Pharmacy, Division of Medicinal Chemistry and Natural Products Chapel Hill, North Carolina 27599-7360

FIGURES

Figure 1. Chromosome end replication must overcome two problems. Lagging strand DNA synthesis does not complete the replication of the 5' end resulting in a 3' overhang, which could contribute to loss of telomeric DNA. Complete leading strand DNA synthesis results in a blunt end though the mature structure contains a 3' overhang. Parental DNA is indicated as either red or blue, and replicated DNA is black. Arrows show the direction of the replication fork.

Figure 2. The telomere forms a lasso-like loop structure under the direction of the shelterin complex. A. Six proteins, TRF 1, TRF2, TPP1, POT1, TIN2, and RAP1 form a dedicated telomere-protection protein complex in humans. B. Human telomeres end in a t-loop characterized by invasion of the 3' overhang into a region of double stranded telomeric DNA. C. Shelterin caps the telomere in part by inducing t-loop formation.

Figure 3. The t-loop can be tied with three different knots. A. The 3' overhang inserts into the double stranded DNA to form a new duplex and a displacement loop. B. The strand invasion structure resembles a Holliday junction after branch migration of the 3' overhang insertion point. C. The 3' overhang associates with the G-strand of the insertion point via G-quadruplexes. The C-strand could either be single stranded or form an I-motif.

Figure 4. Domain structure of hTERT and hTR. Features of the domain structure of hTERT include the RT motifs (1, 2, A, B', C, D, and E) and telomerase specific T- GQ, CP, and QFP motifs, which were identified by sequence analysis. The RNA interaction domain 1 (RID1), N-terminal dissociates activities of telomerase (N-DAT), and RNA interaction domain 2 (RID2) domains were defined functionally. Positions that have been identified as hTERT binding sites are indicated. Domains of hTR that are conserved among vertebrates are indicated.

Figure 5. Solution structures of human telomerase RNA domains. Black dashed lines indicate the location of the individual structures in the full-length RNA. The full-length RNA and sequence of the individual structure elements are colored to match the ribbon diagrams. For the pseudoknot, the p2b helix is cyan, the p3 helix is green, the J2a/3 loop is blue, the J2b/3 loop is pink, and U177, which was deleted in the construct used for structure determination is black. For the CR4/CR5 domain, the p6a helix is cyan, the J6 bulge is green, the p6b helix is pink, the p6.1 helix is cyan, uridines in the loop are orange and guanosines in the loop are green. Residues in the NMR structures that are not native to human TR are colored grey. Coordinates for all structures were rendered using PyMol (www.pymol.org).

Figure 6. G-quadruplex structures. A. The chemical structure of the G-quartet forms a network of Hoogsteen base pairs with a monovalent cation at its core. B. DNA with runs of guanosine rich sequences can form several quartets that stack to form a variety of G-quadruplex structures. A single DNA strand can fold upon itself to form an intramolecular G-quadruplex, while two and four DNA strands can assemble into dimeric and tetrameric structures, respectively. The arrows indicate the parallel or anti-parallel orientation of DNA strands. Guanosine residues are colored according to the configuration of the glycosidic bind. Anti guanosines are colored aqua and syn guanosines are colored orange.

Figure 7. The structures observed for the intramolecular quadruplex formed by the human telomeric repeat sequence. A. The solution structure in Na⁺. B. The crystal structure in the presence of K⁺. C. The solution structure in K⁺. Schematic structures are colored as in Figure 6.

Figure 8. The relationship between telomere length and cellular aging. Telomere length, represented on the ordinate, is progressively lost during successive rounds of cellular replication, represented on the abscissa, in telomerase negative somatic cell lines. This leads to Rb- and p53- mediated growth arrest, marked as senescence. Inactivation of p53 and Rb function bypasses senescence leading to further cell growth and further telomere erosion. Telomere shortening leads to genetic instability and crisis. Rare survivors reactivate a telomere-stabilization pathway, either telomerase or ALT. Germline cells are telomerase positive and maintain the lengths of their telomeres. Pluripotent stem cells express some telomerase activity, but not enough to fully maintain the full length.

Figure 9. Representative telomerase-targeted inhibitors. BIBR 1532 was identified by high throughput screening followed by optimization of lead compounds. UCS1025A is a natural product isolated from the fungus Acremonium sp. GRN 163L is a telomerase template antagonist that is a short oligonucleotide that is complementary to the telomerase template. GRN163L contains a unique N3'® P5' thio-phosphoramidate backbone.

Figure 10. G-quadruplex binding ligands. Telomestatin is natural product isolated from Streptomyces amulatus. BRACO 19 is a 3,6,9 trisubstituted acridine derivative. RHPS4 is a pentacyclic acridine derivative. 115405 is a trisubstituted triazine derivative.