Development of a T-Loop Assay to Investigate T-Loop Dynamics and Structure
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An attractive target in cancer therapy development has been the study of telomeres, which are repetitive sequences at the ends of chromosomes critical in maintaining genomic stability. T-loops, formed by the 3’ overhang inserting into the double strand region of the telomere, are thought to protect the ends from being recognized as double-strand breaks. An almost universal marker for cancer, telomerase, is a promising therapeutic target since its inhibition results in critically short telomeres, compromising t-loop structures. The clinical application has yet to be fully realized due to the lag phase between the times in which telomerase is inhibited and the times that telomeres become sufficiently short that the tumor undergoes apoptosis. Thus, improvements are needed including a greater understanding in t-loop dynamics and the cooperative interaction with telomerase to provide the strategy in which the lag phase can be shortened. Unfortunately, the study of t-loops has been challenging due to the difficulty of isolating and visualizing DNA with intact loop structures. Thus, we have developed novel methods to isolate DNA such that biochemical assays and microscopic visualizations for authentic t-loops are now possible. Digestion of proteins that stabilize t-loop and significant melting at the ends of DNA allow the 3’overhang to migrate out of the double strand region, thus unfolding t-loop and exposing the overhang. DNA isolated with typical procedures (Proteinase K at 55°C for 4 hrs, phenol/chloroform extraction) are thus linear and telomeric overhangs are susceptible to digestion by a 3’-5’ exonuclease (ExoI). The “overhangs” in t-loop structures should be resistant to ExoI. If we lower the temperature of Proteinase K digestion to 4°C to reduce the amount of DNA melting that can occur, we find that the ends are in fact resistant to ExoI digestion. A consistent ~2 fold higher overhang signal in isolated t-loops compared to linear telomeres was observed to distinguish between the two samples. Heating these 4°C samples to 37°C and 55°C caused unfolding of t-loops, resulting in sensitivity of the overhangs to ExoI to the same extent as normal DNA preparations. To validate the t-loop assay, transmission electron microscopy (TEM), a method with powerful magnification and extremely high resolution, is used to visualize DNA isolated at 55°C (linear structures) and 4°C (t-loop structures). The assay was then used to investigate t-loop dynamics throughout cell cycle, and we found that t-loops remain in a folded conformation throughout S phase, confirming the hypothesis that t-loops would unfold a second time for late S/G2 C-strand fill in. We also found that an overhang size of ~30 nts is too short to maintain stabilized t-loops compared to ~90 nts in BJ cells. In summary, we have significant evidence that we are able to prepare and analyze t-loops. Using this assay to determine t-loop structure and timing of t-loop repackage following replication and telomerase action will exceedingly add to our understanding of telomere biology. These are the key steps in setting the stage for many additional future studies, such as what factors contribute in generation of t-loops, how t-loop folding varies with telomere length, and what is the timing of t-loop folding and unfolding throughout cell cycle. All of which will provide critical information for the discovery of new improvements in anti-telomerase therapeutics.