2D gel electrophoresis reveals dynamics of t-loop formation during the cell cycle and t-loop in maintenance regulated by heterochromatin state [DNA and Chromosomes]

Linear chromosome ends are capped by telomeres that have been previously reported to adopt a t-loop structure. The lack of simple methods for detecting t-loops has hindered progress in understanding the dynamics of t-loop formation and its function in protecting chromosome ends. Here, we employed a classical two-dimensional agarose gel method (2D gel method) to innovatively apply to t-loop detection. Briefly, restriction fragments of genomic DNA were separated in a 2D gel, and the telomere sequence was detected by in-gel hybridization with telomeric probe. Using this method, we found that t-loops are present throughout the cell cycle and t-loop formation tightly couples to telomere replication. We also observed that t-loop abundance positively correlates with chromatin condensation, i.e. cells with less compact telomeric chromatin (ALT cells and trichostatin A [TSA]-TSA treated HeLa cells) exhibited fewer t-loops. Moreover, we observed that telomere dysfunction-induced foci (TIFs), ALT-associated PML bodies (APBs), and telomere sister chromatid exchanges (T-SCEs) are activated upon TSA-induced loss of t-loops. These findings confirm the importance of the t-loop in protecting linear chromosomes from damage or illegitimate recombination.

from ! Human Diseases via Alexandros G.Sfakianakis on Inoreader https://ift.tt/2SyMRxk