Telomere Terminal G/C Strand Synthesis: Measuring Telomerase Action and C-Rich Fill-In.
Telomerase is present in most human cancers, and proliferative stem cells including germline cells. Telomerase plays an essential role in tumorigenesis by maintaining/elongating telomeric DNA, and thus preventing the telomere shortening that results in replicative senescence. Understanding telomerase action in vivo has important implication for both cancer and aging, but there are not robust methods for monitoring telomerase action. By combining a series of cell biological and biochemical approaches, and taking advantage of the enzyme DSN that specifically cuts double-stranded DNA and releases the telomeric overhangs, we have developed a method to monitor telomerase action during one cell cycle. Here, we describe this method using Hela carcinoma cells as an example.
Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.
Yong
Y
Zhao
Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA.
Jerry W
JW
Shay
Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390, USA. woodring.wright@utsouthwestern.edu.
Woodring E
WE
Wright
D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D016922:Cellular Senescence / Q000235:genetics; D004247:DNA / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D019098:Telomerase / Q000235:genetics; D016615:Telomere / Q000235:genetics*
D004247:DNA; D019098:Telomerase
BrdU; C-rich fill-in; Cell synchronization; CsCl gradients; Duplex Specific Nuclease (DSN); Overhang; Telomerase; Telomere