Terace M. Fletcher

The effect of the TRF2 N-terminal and TRFH regions on telomeric G-quadruplex structures

The sequence of human telomeric DNA consists of tandem repeats of 5′-d(TTAGGG)-3′. This guanine-rich DNA can form G-quadruplex secondary structures which may affect telomere maintenance. A current model for telomere protection by the telomere-binding protein, TRF2, involves the formation of a t-loop which is stabilized by a strand invasion-like reaction. This type of reaction may be affected by G-quadruplex structures. We analyzed the influence of the arginine-rich, TRF2 N-terminus (TRF2B), as well as this region plus the TRFH domain of TRF2 (TRF2BH), on the structure of G-quadruplexes. Circular dichroism results suggest that oligonucleotides with 4, 7 and 8 5′-d(TTAGGG)-3′ repeats form hybrid structures, a mix of parallel/antiparallel strand orientation, in K+. TRF2B stimulated the formation of parallel-stranded structures and, in some cases, intermolecular structures. TRF2BH also stimulated intermolecular but not parallel-stranded structures. Only full-length TRF2 and TRF2BH stimulated uptake of a telomeric single-stranded oligonucleotide into a plasmid containing telomeric DNA in the presence of K+. The results in this study suggest that G-quadruplex formation inhibits oligonucleotide uptake into the plasmid, but the inhibition can be overcome by TRF2. This study is the first analysis of the effects of TRF2 domains on G-quadruplex structures and has implications for the role of G-quadruplexes and TRF2 in the formation of t-loops.

Stress-induced senescence exaggerates postinjury neointimal formation in the old vasculature.

This study aims to demonstrate the role of stress-induced senescence in aged-related neointimal formation. We demonstrated that aging increases senescence-associated beta-galactosidase activity (SA-beta-Gal) after vascular injury and the subsequent neointimal formation (neointima-to-media ratio: 0.8 +/- 0.2 vs. 0.54 +/- 0.15) in rats. We found that senescent cells (SA-beta-Gal(+) p21(+)) were scattered throughout the media and adventitia of the vascular wall at day 7 after injury and reached their maximum number at day 14. However, senescent cells only persisted in the injured arteries of aged animals until day 30. No senescent cells were observed in the noninjured, contralateral artery. Interestingly, vascular senescent cells accumulated genomic 8-oxo-7,8-dihydrodeoxyguanine, indicating that these cells were under intense oxidative stress. To demonstrate whether senescence worsens intimal hyperplasia after injury, we seeded matrigel-embedded senescent and nonsenescent vascular smooth muscle cells around injured vessels. The neointima was thicker in arteries treated with senescent cells with respect to those that received normal cells (neointima-to-media ratio: 0.41 +/- 0.105 vs. 0.26 +/- 0.04). In conclusion, these results demonstrate that vascular senescence is not only a consequence of postinjury oxidative stress but is also a worsening factor for neointimal development in the aging vasculature.

Telomerase: a potential therapeutic target for cancer

Telomeres are complex structures which serve to protect chromosome ends. Telomere shortening occurs in normal somatic cells reaching a point in which cells senesce. Senescence can be counteracted by activating telomerase. Telomerase activity is present in a majority of cancer cells and requires the upregulation of the reverse transcriptase component called hTERT. Because telomerase activity is essential for proliferation of most cancer cells, therapeutic strategies have been developed to inhibit its activity. These strategies centre on targeting the active site, hTERT and hTERC expression, core enzyme stability and telomeric DNA. Successful approaches involve a combination of traditional drugs with telomerase inhibitors. Disrupting the functional expression of hTERT is particularly effective in agreement with evidence that hTERT is an antiapoptotic factor in some cancer cells. In addition, approaches that stabilise DNA secondary structures may disrupt telomere maintenance through a variety of routes making them, potentially, very potent in attack-ing cancer cells.

The telomere binding protein trf2 induces chromatin compaction

Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

The Myb/SANT domain of the telomere-binding protein TRF2 alters chromatin structure

Eukaryotic DNA is packaged into chromatin, which regulates genome activities such as telomere maintenance. This study focuses on the interactions of a myb/SANT DNA-binding domain from the telomere-binding protein, TRF2, with reconstituted telomeric nucleosomal array fibers. Biophysical characteristics of the factor-bound nucleosomal arrays were determined by analytical agarose gel electrophoresis (AAGE) and single molecules were visualized by atomic force microscopy (AFM). The TRF2 DNA-binding domain (TRF2 DBD) neutralized more negative charge on the surface of nucleosomal arrays than histone-free DNA. Binding of TRF2 DBD at lower concentrations increased the radius and conformational flexibility, suggesting a distortion of the fiber structure. Additional loading of TRF2 DBD onto the nucleosomal arrays reduced the flexibility and strongly blocked access of micrococcal nuclease as contour lengths shortened, consistent with formation of a unique, more compact higher-order structure. Mirroring the structural results, TRF2 DBD stimulated a strand invasion-like reaction, associated with telomeric t-loops, at lower concentrations while inhibiting the reaction at higher concentrations. Full-length TRF2 was even more effective at stimulating this reaction. The TRF2 DBD had less effect on histone-free DNA structure and did not stimulate the t-loop reaction with this substrate, highlighting the influence of chromatin structure on the activities of DNA-binding proteins.

Telomere Higher‐Order Structure and Genomic Instability

Telomeres, nucleoprotein complexes at the end of eukaryotic chromosomes, have vital roles in chromosome integrity. Telomere chromatin structure is both intricate and dynamic allowing for a variety of responses to several stimuli. A critical determinant in telomere structure is the G‐strand overhang. Facilitated by telomeric proteins, the G‐strand overhang stabilizes telomere higher‐order assemblies most likely by adopting unusual DNA structures. These structures influence activities that occur at the chromosome end. Dysfunctional telomeres induce signals resulting in cell growth arrest or death. To overcome telomere dysfunction, cancer cells activate the DNA polymerase, telomerase. The presence of telomerase at the telomere may establish a particular telomeric state. If the chromosome ends of cancer and normal cells exist in different states, cancer‐specific telomere structures would offer a unique chemotherapeutic target. IUBMB Life, 55: 443‐449, 2003