A DNA-fiber protocol for single molecule analysis of telomere (SMAT) length and extension events in cancer cells

Distinct modes of telomere synthesis and extension contribute to Alternative Lengthening of Telomeres

“…To characterize the effects of cell-cycle arrest directly on telomere extension, we employed single molecule analysis of telomeres (SMAT), which utilizes molecular combing of telomere fibers. 17 Cells were labeled with EdU for 2 h (i) as cells cycled from G2 to M (10 h post-thymidine release), (ii) during G2 arrest (G2+8 h), (iii) during mitotic release from G2 arrest (G2+6 h M), or (iv) during mitotic arrest (M+4 h, M+14 h) ( Figures 1 A and 2 B). Notably, we discovered a lack of detectable telomere extension events, defined by EdU tracts at the termini of telomere fibers, in cells labeled during G2 arrest and during mitotic arrest ( Figures 2 A and 2B).…”

“…DNA fibers, stained for telomeres, DNA, and labeled for EdU, were prepared as described previously for U-2 OS, HeLa LT and GM847 cells. 17 SMAT was performed on EdU-labelled G2 arrested cells and mitotic cells (selected via mitotic shake-off) released for 2 h from G2 arrest and cells labeled for 2 h during G2/M defined as 9–11 h post-thymidine release. G2 arrested cells were labeled for the last 2 h prior to the indicated timepoint.…”

“…Here, we combine cell cycle synchronization with single molecule analysis of telomeres (SMAT) performed on stretched DNA fibers 14 to precisely measure and temporally track ALT-mediated telomere synthesis events through the cell cycle. We identify cell cycle-associated modes of non-productive and productive telomere synthesis in ALT cells.…”

Deregulated DNA ADP-ribosylation impairs telomere replication

“…SMAT replication was performed as described 50 . Briefly, cells were labeled with 10 μM EdU for 5 h before collection by trypsinization.…”

Techniques for assessing telomere length: A methodological review

“…It is also expected that TCA may further be improved by using chromosome-specific subtelomere probes enabling measurement of chromosome specific telomere lengths [56] . This method's ability to directly visualize telomeres offers insights into the heterogeneity of telomere lengths, enriching our understanding of the mechanisms behind aging and disease processes [57] , [58] .…”