IFOM ETS-The AIRC Institute of Molecular Oncology, Milan, Italy.
Institute for Tumor Biology and Experimental Therapy, Frankfurt Cancer Institute, Goethe University Frankfurt, Frankfurt am Main, Germany.
Sci Adv. 2023 Mar 22;9(12):eadf2011. doi: 10.1126/sciadv.adf2011.
The telomerase reverse transcriptase elongates telomeres to prevent replicative senescence. This process requires exposure of the 3'-end, which is thought to occur when two sister telomeres are generated at replication completion. Using two-dimensional agarose gel electrophoresis (2D-gels) and electron microscopy, we found that telomeric repeats are hotspots for replication fork reversal. Fork reversal generates 3' telomeric ends before replication completion. To verify whether these ends are elongated by telomerase, we probed de novo telomeric synthesis in situ and at replication intermediates by reconstituting mutant telomerase that adds a variant telomere sequence. We found variant telomeric repeats overlapping with telomeric reversed forks in 2D-gels, but not with normal forks, nontelomeric reversed forks, or telomeric reversed forks with a C-rich 3'-end. Our results define reversed telomeric forks as a substrate of telomerase during replication.
端粒酶逆转录酶通过延长端粒来防止复制性衰老。这一过程需要暴露 3'端，人们认为这发生在复制完成时产生两个姐妹端粒的时候。通过二维琼脂糖凝胶电泳（2D-gels）和电子显微镜，我们发现端粒重复序列是复制叉反转的热点。叉反转在复制完成之前产生 3'端粒末端。为了验证这些末端是否通过端粒酶延长，我们通过重新构建添加变体端粒序列的突变端粒酶，原位探测新合成的端粒和复制中间体。我们发现，在 2D 凝胶中，变体端粒重复序列与复制叉反转重叠，但与正常叉、非端粒反转叉或 3'端富含 C 的端粒反转叉不重叠。我们的结果将反转的端粒叉定义为复制过程中端粒酶的底物。
