As DNA strands ravel and unravel in an intricate dance, one notable event takes center stage: replication. This process is essential to life, but the finer details of its orchestrated steps are still being uncovered. During replication, the ends of linear DNA cannot fully replicate, causing telomeres to shorten in a well-known phenomenon called the end-replication problem.1
The discovery of telomerase in the 1980s appeared to solve this problem, where naturally occurring telomerase added single-stranded G-rich TTAGGG repeats to the telomere ends.2 However, Titia de Lange, a cell biologist and geneticist at Rockefeller University, and her team realized that it was only part of the solution. Their findings, published recently in Nature, demonstrated that while telomerase addressed the end-replication problem at the leading strand, another complex worked in tandem at the lagging strand.3 This provides insights into telomere biology in health and disease.
Hiroyuki Takai, a cell biologist and geneticist in de Lange’s group, made the unexpected discovery while studying the lagging strand in cells that lacked a molecular complex comprised of a protein group, CTC1–STN1–TEN1 (CST), bound to polymerase α-primase (Polα-primase). CST-Polα-primase is telomerase’s counterpart on the lagging strand that adds C-rich CCCTAA repeats to telomere ends. However, when Takai studied telomeres in cells lacking the CTC1 gene, which disabled CST-Polα-primase, he produced a gel that did not fit the established model. The expected fill-in synthesis was incomplete. “Regular replication of the telomere was supposed to keep up with telomerase and elongate during DNA replication, but it was clear that this was not the case,” remarked de Lange.
In Takai’s experiments, DNA replication proceeded where the leading strand synthesis created a blunt end and telomerase elongated the G strand as expected; however, the fill-in synthesis of the C-rich strand via short DNA fragments (Okazaki fragments) surprisingly could not keep up in these cells. “Not only did it not elongate, but it also got shorter,” said de Lange.
Normally for the lagging strand, DNA synthesis is unhindered, and the replisome starts synthesizing the last Okazaki fragments along the 3’ overhang that are later stitched together to prevent sequence loss. However, nobody knew exactly what happened to the replisome once it reached the end of the DNA. Since Takai’s findings suggested that the replisome fell short of the last Okazaki fragment and led to inadequate primer synthesis along the lagging strand, it was worth investigating. Perplexed, de Lange contacted Joseph Yeeles, a biochemist at the Medical Research Council Laboratory of Molecular Biology, to take a closer look at the replisome during this event.
To study DNA replication in vitro, Yeeles assembled replisomes with Saccharomyces cerevisiae proteins capable of performing complete leading- and lagging-strand replication. He observed that the replisomes initiated Okazaki fragments within 150-200 nucleotides of the ends of the templates but stopped short around 20 nucleotides from the ends of the leading-strand templates. “[The replisome] just can’t do it. It cannot get its foot on the last bit of DNA before falling off,” remarked de Lange. This shortening of the lagging-end telomeres supported the existence of a second end-replication problem.
Takai sought to confirm Yeeles’ findings in vivo. Previous work by de Lange’s team and others explored the effect of disabling CST-Polα-primase; however, the rate and cause of shortening at the telomeric sites remained unclear.4 Takai isolated and measured sequence loss at the leading- and lagging-end telomeres in cells that lacked CTC1. By using a cesium chloride gradient, Takai saw results that echoed Yeeles’ in vitro analysis: the G strand grew while the C strand shortened. This reinforced the inability of the lagging strand to reach the same length as its counterpart.
Telomerase prevented shortening at the leading strand while CST-Polα-primase addressed the same problem at the lagging strand. With CST-Polα-primase, the complex counteracted the loss of approximately 76 nucleotides in the C-strand, underscoring its role in telomere biology. “This study highlights a second telomere maintenance machinery,” remarked Ci Ji Lim, a molecular cell biologist at the University of Wisconsin-Madison who was not involved in the study. “It’s a watershed announcement that presents a bird’s eye view and appreciation for not just telomerase, but for other less known proteins like CST-Polα-primase.”
“This is great for the field because there’s a framework where researchers can fill in the molecular details. So, I expect that we’ll learn a lot more about C strand fill-in down the road,” remarked Lim.
de Lange and her team aim to further explore the structural biology of key regulators of telomerase and identify the kinase critical for end replication. They hope that these findings also provide insight into clinical implications for individuals with telomere disorders.
- Lingner J, et al. Telomerase and DNA end replication: no longer a lagging strand problem? Science. 1995;269(5230):1533-1534.
- Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 1985;43(2 Pt 1):405-413.
- Takai H, et al. CST–polymerase α-primase solves a second telomere end-replication problem. Nature. 2024;627:664–670.
- Takai H, et al. A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus. Genes Dev. 2016;30(7):812-826.
