G-quadruplex DNA and the Regulation of Human Telomere Accessibility

Human telomeres are nucleoprotein structures that cap the ends of chromosomes, preventing them from degradation. In normal cells, they are shortened due to the end replication problem. However, most cancer cells overcome this inherent growth limitation by upregulating telomerase, an enzyme that elongates telomeres to yield an immortal phenotype. The remaining cancerous cells immortalize by activating the alternative telomerelengthening pathway (ALT). Thus, treatments that directly target the telomere could disrupt both mechanisms the cancer cell employs for unlimited proliferation. The telomere overhang is G-rich DNA that spontaneously folds into a G-quadruplex. In vitro, the G-quadruplex structure has been shown to inhibit telomere extension by telomerase, making it an attractive potential therapeutic target. However, G-quadruplex capping properties and regulatory roles are still poorly understood. In this study, we used a FRET-based assay to probe the stability and accessibility of the G-quadruplex. We examined the folding behavior of variable telomeric repeat lengths (4- 8). Accessibility of the G-quadruplex was measured by the rate of quadruplex unfolding after the addition of complementary DNA, ALT pathway proteins, and telomerase. These measurements revealed a distinct periodic pattern of overhang accessibility where DNA and protein binding is limited for multiples of four TTAGGG repeats, whereas five to seven repeats are more accessible (7>6>5). Contrastingly, POT1, a telomere-specific binding protein, showed accessibility independent of the number of repeats. These results demonstrate the role of G-quadruplexes as physical impedances to the binding of telomere associated proteins and as critical regulatory structures for theOpe